Multi-stage personalized longevity therapeutics

ABSTRACT

A method of treating senescence in a subject can include applying a senoremediation drug treatment protocol to the subject in order to rescue one or more first cells in the subject, wherein the senoremediation drug treatment protocol is derived from a computational transcriptome analysis of the tissue or organ of the subject. The method can include applying a senolytic drug treatment protocol to the subject in order to remove one or more second cells in the subject. The method can include introducing stem cells into a tissue and/or organ of the subject in order to rejuvenate one or more tissue cells in the tissue and/or one or more organ cells in the organ. The method can include carrying out a reinforcement step that includes one or more actions that prevent further senescence or degradation of the tissue or organ.

CROSS-REFERENCE

This patent application claims priority to U.S. Provisional ApplicationNo. 62/536,658 filed Jul. 25, 2017, which provisional is incorporatedherein by specific reference in its entirety.

This patent application claims priority to U.S. Provisional ApplicationNo. 62/547,061 filed Aug. 17, 2017, which provisional is incorporatedherein by specific reference in its entirety.

BACKGROUND

Until recently, treatments and therapies for senescence reversal (agingreversal) have been rare, largely because of the complexity of theunderlying mechanisms of senescence and the lack of tools forunderstanding and treating senescence. One example of drug developmentfor senescence protection (rather than senescence reversal) can be seenin US 2017/0073735. Recent bioinformatics developments such as deepneural networks have opened up the possibility of developinghighly-personalized senescence reversal treatments, based on geneexpression of senescent tissues versus non-senescent tissues, as will bedisclosed in the present invention.

While aging may be a complex multifactorial process with no single causeor treatment, the issue whether aging can be classified as the diseaseis widely debated. Many strategies for extending organismal life spanshave been proposed including replacing cells and organs, comprehensivestrategies for repairing the accumulated damage, using hormetins toactivate endogenous repair processes, modulating the aging processesthrough specific mutations, gene therapy and small molecule drugs. Ananimal's survival strongly depends on its ability to maintainhomeostasis, achieved partly through intracellular and intercellularcommunication within and among different tissues.

Presently, none of the proposed strategies for senescence treatmentprovide a roadmap for rapid screening, validation and clinicaldeployment. No methods currently exist to predict the effects ofcurrently available drugs on human longevity and health span in a timelymanner.

Many biomarkers of aging have been proposed including telomere length,intracellular and extracellular aggregates, racemization of the aminoacids and genetic instability. Gene expression and DNA methylationprofiles change during aging also may be used as biomarkers of aging.Many studies analyzing transcriptomes of biopsies in a variety ofdiseases indicated that age and sex of the patient have significanteffects on gene expression and that there are noticeable changes in geneexpression with age in mice, resulting in development of mouse aginggene expression databases and in humans.

Combinations of protein-protein interaction and gene expression in bothflies and humans demonstrate that aging is mainly associated with asmall number of biological processes, which might preferentially attackkey regulatory nodes that are important for network stability.

Work of the inventors, among others, with gene expression andepigenetics of various solid tumors provided clues that transcriptionprofiles of cells mapped onto the signaling pathways may be used toscreen for and rate the targeted drugs that regulate pathways directlyand indirectly related to aging and longevity. Prior studies suggestthat a combination of pathways, termed pathway cloud, instead of oneelement of the pathway or the whole pathway might be responsible forpathological changes in the cell.

The senescence response causes striking changes in cellular phenotype.Aging/senescence in humans causes striking changes in cellularphenotype. According to (Campisi and d'Adda di Fagagna 2007) thesenescent phenotype is induced by multiple stimuli. Mitoticallycompetent cells respond to various stressors by undergoing cellularsenescence. These stressors include dysfunctional telomeres,non-telomeric DNA damage, excessive mitogenic signals including thoseproduced by oncogenes (which also cause DNA damage), non-genotoxicstress such as perturbations to chromatin organization and, probably,stresses with an as-yet unknown etiology. These changes include anessentially permanent arrest of cell proliferation, development ofresistance to apoptosis (the death of some cells that occurs as a normaland controlled part of an organism's growth or development) and analtered pattern of gene expression. Also, the expression or appearanceof senescence-associated markers such as senescence-associatedβ-galactosidase, p16, senescence-associated DNA-damage foci (SDFs) andsenescence-associated heterochromatin foci (SAHFs) are neither universalnor exclusive to the senescent state.

Cellular senescence is thought to contribute to age-related tissue andorgan dysfunction and various chronic age-related diseases throughvarious mechanisms. Senescence is characterized by a persistentproliferative arrest in which cells display a distinct pro-inflammatorysenescent-associated secretory phenotype (SASP) (Krimpenfort and Berns2017). Whereas SASP exerts a supportive paracrine function during earlydevelopment and wound healing (Demaria et al. 2014), the continuoussecretion of these SASP factors has detrimental effects on normal tissuehomeostasis and is considered to significantly contribute to aging(DiLoreto and Murphy 2015).

In a cell-autonomous manner, senescence acts to deplete the variouspools of cycling cells in an organism, including stem and progenitorcells. In this way, senescence interferes with tissue homeostasis andregeneration, and lays the groundwork for its cell-non-autonomousdetrimental actions involving the SASP. There are at least five distinctparacrine mechanisms by which senescent cells are thought to promotetissue dysfunction, including perturbation of the stem cell niche(causing stem cell dysfunction), disruption of extracellular matrix,induction of aberrant cell differentiation (both creating abnormaltissue architecture), stimulation of sterile tissue inflammation, andinduction of senescence in neighboring cells (paracrine senescence). Anemerging yet untested concept is that post-mitotic, terminallydifferentiated cells that develop key properties of senescent cellsmight contribute to ageing and age-related disease through the same setof paracrine mechanisms (van Deursen 2014).

Several recent observations support the hypothesis that senescence is ahighly-dynamic, multi-step process, during which the properties ofsenescent cells continuously evolve and diversify, much liketumorigenesis but without cell proliferation as a driver (De Cecco etal. 2013; Wang et al. 2011; Ivanov et al. 2013). This includes not onlysenescent cells but also take in account pre-senescent stage. This factalso means there is an opportunity to reverse the cell to normalnon-senescent behavior.

At least two general concepts of age exist in the art. One,“chronological age” is simply the actual calendar time an organism orhuman has been alive. Another one, called “biological age” or“physiological age”, which is a particular focus of the presentinvention, is related to the physiological health of the individual, andbiomarkers thereof. Biological age is associated with how well organsand regulatory systems of the body are performing and at what extent thegeneral homeostasis at all levels of the organism is being maintained,as such functions generally decline with time and age.

The measurement of any physiological process of an organism is typicallydone with a set of predefined biomarkers. A biomarker can be defined asa characteristic that is objectively measured and evaluated as anindicator of normal biological processes, pathogenic processes, orpharmacologic responses to a therapeutic intervention. Biomarkers arechosen by scientists in order to measure a very-well defined processwithin the body.

Given that in a multi-cellular organism that aging is a systemicprocess, which cannot be readily captured by single uni-dimensional oreven several metrics, the development of an accurate and useful measureof biological age (which can be thought of as a biological clock), issubject to specific challenges. Again, such biomarkers must not only bean objective quantifiable and easily measurable characteristics of thebiological aging process, but must also be able to take into accountthat aging is not a single specific process, but rather a suite ofchanges across multiple physiological systems.

In other words, no single biomarker can provide an accurate overallbiological clock age of a multi-cellular organism, nor can thebiological age of a single cell, tissue, or organ, even when composed ofmany biomarkers, provide an accurate overall biological age of anorganism. And in fact, it is often useful to have several biologicalclocks assigned to an organism or human, that is, a different biologicalage can be assigned to different cells, tissues, or organs of thatorganism, as well as different clocks based on a different biomarker ordifferent biomarker. Thus there may be one clock for the skin, one forthe liver, one clock based on telomere length of a cell(s), tissue(s),or organ(s), and another based on a different biomarker.

In the past, several attempts have been made to develop adaptedbiomarkers for measuring biological aging. However, the biomarkers usedso far focus on monitoring a restricted number of processes known forbeing directly involved in the onset and propagation of aging relateddamages through the body. Examples of such biomarkers are telomerelength (Lehmann, 2013), intracellular and extracellular aggregates,racemization of the amino acids and genetic instability. Both geneexpression (Wolters, 2013) and DNA methylation profiles (Horvath, 2012,Horvath, 2013, Mendelsohn, 2013) change during aging and may be used asbiomarkers of aging as demonstrated previously with the epigenetic clock(Horvath, 2012, Horvath, 2013). Many studies analyzing transcriptomes ofbiopsies in a variety of diseases indicated that age and sex of thepatient had significant effects on gene expression (Chowers, 2003) andthat there are noticeable changes in gene expression with age in mice(Weindruch, 2002, Park, 2009), resulting in development of mouse aginggene expression databases (Zahn, 2007) and in humans (Blalock, 2003;Welle, 2003; Park, 2005; Hong, 2008; de Magalhães, J. P, 2009).

Work of the inventors, among others, with gene expression andepigenetics of various solid tumors provided clues that transcriptionprofiles of cells mapped onto the signaling pathways may be used toscreen for and rate the targeted drugs that regulate pathways directlyand indirectly related to aging and longevity. Prior studies suggestedthat a combination of pathways, termed pathway cloud, instead of oneelement of the pathway or the whole pathway might be responsible forpathological changes in the cell.

There has always been a need to reverse senescence, but only recentlyare there the necessary tools, particularly, developments in informaticsand machine learning, to develop and apply such senescence therapies andtreatments. Further, even commonly-accepted biomarkers and metric ofsuch biomarkers to assess aging have been lacking.

SUMMARY

In some embodiments, a method of treating senescence in a subject caninclude applying a senoremediation drug treatment protocol to thesubject in order to rescue one or more first cells in the subject,wherein the senoremediation drug treatment protocol is derived from acomputational transcriptome analysis of the tissue or organ of thesubject. In some aspects, the method can include applying a senolyticdrug treatment protocol to the subject in order to remove one or moresecond cells in the subject. In some aspects, the senolytic drugtreatment protocol is derived from the computational transcriptomeanalysis of the tissue or organ of the subject. In some aspects, themethod can include introducing stem cells into a tissue and/or organ ofthe subject in order to rejuvenate one or more tissue cells in thetissue and/or one or more organ cells in the organ. In some aspects, atleast one of the following is derived from a computational transcriptomeanalysis of the tissue or organ of the subject: identification of thetissue for receiving the stem cells; or identification of the organ forreceiving the stem cells. In some aspects, the method can includecarrying out a reinforcement step that includes one or more actions thatprevent further senescence or degradation of the tissue or organ. Insome aspects, the method can include the one or more actions thatprevent further senescence or degradation of the tissue or organ isderived from the computational transcriptome analysis of the tissue ororgan of the subject. In some aspects, the method can include: applyinga senolytic drug treatment protocol to the subject in order to removeone or more second cells in the subject. In some aspects, the method caninclude introducing stem cells into a tissue and/or organ of the subjectin order to rejuvenate one or more tissue cells in the tissue and/or oneor more organ cells in the organ. In some aspects, at least one of thefollowing is derived from a computational transcriptome analysis of thetissue or organ of the subject: the senolytic drug treatment protocol;identification of the tissue for receiving the stem cells; oridentification of the organ for receiving the stem cells. In someaspects, the method can include carrying out a reinforcement step thatincludes one or more actions that prevent further senescence ordegradation of the tissue or organ. In some aspects, at least one of thefollowing is derived from a computational transcriptome analysis of thetissue or organ of the subject: the senolytic drug treatment protocol;identification of the tissue for receiving the stem cells;identification of the organ for receiving the stem cells; or the one ormore actions that prevent further senescence or degradation of thetissue or organ.

In one embodiment, a method of treating senescence in a subject caninclude: (a) applying a senoremediation drug treatment protocol to thesubject in order to rescue one or more first cells in the subject; (b)applying a senolytic drug treatment protocol to the subject in order toremove one or more second cells in the subject; (c) introducing stemcells into a tissue and/or organ of the subject in order to rejuvenateone or more tissue cells in the tissue and/or one or more organ cells inthe organ; and (d) carrying out a reinforcement step that includes oneor more actions that prevent further senescence or degradation of thetissue or organ. In one aspect, at least one of the following is derivedfrom a computational transcriptome analysis of the tissue or organ ofthe subject: the senoremediation drug treatment protocol; the senolyticdrug treatment protocol; identification of the tissue for receiving thestem cells; identification of the organ for receiving the stem cells;and the one or more actions that prevent further senescence ordegradation of the tissue or organ.

In some aspects, the first cells that are rescued are characterized aspre-senescent cells. In some aspects, the second cells that are removedare characterized as senescent cells. In some aspects, (a) (b) (c) or(d) are carried out in any order or any combination thereof carried outsimultaneously. In some aspects, the method includes repeating at leastone of (a) (b) (c) or (d) at least once. In some aspects, the stem cellsare mesenchymal or epithelial stem cells or both. In some aspects, step(d) further comprises applying a reinforcement drug treatment protocolto the patient. In some aspects, the reinforcement drug treatmentprotocol includes at least one of: immunomodulation, cytoprotection, orstimulation of macrophages. In some aspects, the reinforcement drugtreatment protocol includes at least one of: Insulin receptor substrate(Tyr608) peptide; 740 Y-P; Sapanisertib; Dactolisib; GSK2334470; MP7;Dasantinib; Quercitin; Flavopiridol; Linifanib; Argatroban; Sorafenib;Tucaresol; Methotrexate; Tacrolimus; Curcumin; Lavendustin A; WithaferinA; Sulphorapane. In some aspects, the computational transcriptomeanalysis produces a transcriptome signature that is used as input to amachine learning platform that outputs drug classifications for one ofsteps (a), (b), or (d). In some aspects, the computational transcriptomeanalysis produces a transcriptome signature that is compared to abaseline transcriptome signature that represents a less senescentversion of the subject's tissue or organ. In some aspects, thecomputational transcriptome analysis produces a transcriptome signaturethat is compared to a baseline transcriptome signature that isconstructed from more than one tissue or organ transcriptome signatureof one or more different subjects.

The foregoing summary is illustrative only and is not intended to be inany way limiting. In addition to the illustrative aspects, embodiments,and features described above, further aspects, embodiments, and featureswill become apparent by reference to the drawings and the followingdetailed description.

BRIEF DESCRIPTION OF THE FIGURES

The foregoing and following information as well as other features ofthis disclosure will become more fully apparent from the followingdescription and appended claims, taken in conjunction with theaccompanying drawings. Understanding that these drawings depict onlyseveral embodiments in accordance with the disclosure and are,therefore, not to be considered limiting of its scope, the disclosurewill be described with additional specificity and detail through use ofthe accompanying drawings.

FIG. 1 shows an embodiment of a 5R strategy, which is applied topatients with pre-senescent, senescent or fibrotic conditions.

FIG. 2 shows the general scheme of an iPANDA calculation pipeline.

FIG. 3 provides information for read-outs for senolytic andsenoremediation compound validation.

FIG. 4 shows a Venn diagram of iPANDA signature pathways for each groupof comparison.

FIG. 5 shows the timeline of senolytic drugs validation.

FIG. 6A shows the PrestoBlue results of INSSEN17 treated cells for allfive tested concentration ranges prior (day 4) after (day 5) and twodays after the treatment (day 7).

FIG. 6B shows a favorable concentration-dependent increase incaspase-positive MMC-treated cells up to a concentration of 6.49 μMwhile significantly differing from the mitotic cells.

FIG. 7 illustrates a transcriptomic clock method for biological agingassessment, compatible with the current invention.

FIG. 8 illustrates the delta (difference between assigned (predicted)biological age and actual chronological age) bar plots grouped by ageranges for healthy people based on an exemplary validation set asdescribed.

FIG. 9 illustrates distribution on number of samples by age for healthyindividuals in the validation set.

FIG. 10 illustrates an example epsilon-prediction accuracy for healthyindividuals.

FIG. 11 is a histogram illustrates clustering using t-SNE clusteringalgorithm by age for healthy individuals.

FIG. 12 shows an example computing device 600 (e.g., a computer) thatmay be arranged in some embodiments to perform the methods (or portionsthereof) described herein.

The elements in the figures are arranged in accordance with at least oneof the embodiments described herein, and which arrangement may bemodified in accordance with the disclosure provided herein by one ofordinary skill in the art.

DETAILED DESCRIPTION

In the following detailed description, reference is made to theaccompanying drawings, which form a part hereof In the drawings, similarsymbols typically identify similar components, unless context dictatesotherwise. The illustrative embodiments described in the detaileddescription, drawings, and claims are not meant to be limiting. Otherembodiments may be utilized, and other changes may be made, withoutdeparting from the spirit or scope of the subject matter presentedherein. It will be readily understood that the aspects of the presentdisclosure, as generally described herein, and illustrated in thefigures, can be arranged, substituted, combined, separated, and designedin a wide variety of different configurations, all of which areexplicitly contemplated herein.

The present invention generally relates to a multi-stage therapeutic fortreating senescence (aging) of whole organisms (in particular, humanindividuals), as well as the organism's underlying cellular, tissue, andorgan senescence. Methods and systems for applying such therapeutictreatment, as well as informatics and other tools for developing thetherapeutic treatments, are disclosed. Since disease and senescence areoften associated, the invention is also applicable to treating disease.

The present disclosure provides compositions and methods for 5R (Rescue,Remove, Replenish, Reinforce, Repeat) strategy selectively rescuingpre-senescent cells, removing senescent cells, replenishing andreinforcing by new healthy cells and repeating the procedure wherein thecomposition comprises a group of senolytics and their derivativesthereof. The strategy of 5R may delay aging and/or treat age-relateddisorders especially fibrotic and senofibrotic disorders primarily inlungs and liver.

This 5R method may delay aging and/or treat age-related disordersespecially fibrotic and senofibrotic disorders primarily in lungs, liverand skin. The 5R strategy as described is applied to patients withpre-senescent, senescent, and fibrotic conditions, among others. Drugsto be used include senoremediators, antifibrotic agents, and senolytics.The 5R approach will result in induction of regeneration. Drugrepurposing strategy can be part of the therapy development process oncethe therapy protocols have been designed.

FIG. 1 shows an embodiment of a 5R strategy, which is applied topatients with pre-senescent, senescent or fibrotic conditions. Thefollowing steps can be performed in any method described herein: 1.Single biopsy procedure; 2. Sample preparation; 3.

Microarray, RNA-seq profiles; 4. iPANDA analysis; 5. Aging clockanalysis; 6. Rescue stage procedure; 7. Senoremediator treatment; 8.Remove stage procedure; 9. Senolytics treatment; 10. Replenish stageprocedure; 11. Therapy with stem cells; 12. Reinforce stage procedure;13. Immunomodulator treatment; and 14. Repeat stage procedure. Any oneof these steps may be performed alone or in combination of other stepsas recited herein. In some instances, the methods can include obtainingdata and processing the data to obtain a recommendation for a treatmentprotocol. The recommended treatment protocol can then be implemented onthe patient in accordance with parameters of the treatment protocol.That is, without the computational generation of the treatment protocol,the aspects of the treatment protocol cannot be performed without theinstructions to do so. As such, obtaining the instructions, such as thetype of drug or specific drug or combination of drugs, can be vital forperforming the treatment protocol.

In some instances, the treatment protocol can be obtained by steps 1, 2,3, 4, and/or 5. Some of these steps may be omitted, such as steps 1, 2,and 3 when the sample is obtained already prepared. In some instancesthe data from 3 may be obtained and provided into a computing system forstep 4 and/or 5. From the data of step 4 and/or 5, steps 6, 7, 8, 9, 10,11, 12, and/or 13 can be performed as determined and in any order or inany combination and with any step being repeated in any order. In oneexample, the method can first perform 6 and then 8, or 8 and then 6.Steps 7 and/or 9 may be performed in any order with respect to eachother. In an example, steps 8 and/or 9 can be performed prior to steps10, 11, 12, or 13.

In some instances, there is a step 4a and/or step 5a, wherein adetermined treatment protocol is provided by step 4 and/or step 5,respectively. The determined treatment protocol can include a list ofone or more drugs or treatment actions for each treatment stepsubsequent to steps 4 and/or 5.

The invention includes methods, system, apparatus, computer programproduct, among others, to carry out the invention described herein.

In one embodiment, a method is provided for treating senescence in apatient tissue or organ, the method comprising: (a) carrying out arescue step comprising applying a senoremediation drug treatmentprotocol to the patient; (b) carrying out a removal step comprisingapplying a senolytic drug treatment protocol to the patient; (c)carrying out a replenishment step comprising introducing stem cells in aparticular tissue or organ; and (d) carrying out a reinforcement stepcomprising one or more preventative actions to prevent furthersenescence or degradation of the tissue or organ. These steps may becarried out in any order or repeated. The stem cells may be mesenchymalor epithelial stem cells or both. The method may further includeapplying a reinforcement drug treatment protocol to the patient, using,immunomodulation, cytoprotection, or stimulation of macrophages. In someaspects, wherein (a) (b) (c) or (d) are carried out in any order. Insome aspects, the method includes repeating at least one of (a) (b) (c)or (d) at least once. In some aspects, the stem cells are mesenchymal orepithelial stem cells or both. In some aspects, (c) further comprisesapplying a reinforcement drug treatment protocol to the patient. In someaspects, (d) further comprising at least one of immunomodulation,cytoprotection, or stimulation of macrophages. In some aspects, atranscriptome signature representing tissue or organ senescence is usedto develop at least one of the drugs used. In some aspects, thetranscriptome signature is used as input to a machine learning platformthat outputs drug classifications. In some aspects, the transcriptomesignature is compared to a baseline transcriptome signature thatrepresents a less senescent version of the patient's tissue or organ. Insome aspects, the transcriptome signature is compared to a baselinetranscriptome signature that is constructed from more than one tissue ororgan transcriptome signature.

In some embodiments, a transcriptome signature representing tissue ororgan senescence may be used to develop at least one of the drugs used,the drugs selected from a senoremediation drug, senolytic drug,antifibrotic drug, or preventative action drugs. The transcriptomesignature may be a signaling pathway activation network analysis, whichmay be used as input into a computer, and processed to obtain therelevant data. Such computer processing of the signaling pathway canprovide information regarding the transcriptome signature that can beobtained as output along with information for the senoremediation drug,senolytic drug, antifibrotic drug, or preventative action drugs. Thetranscriptome signature may be used in the following manner: as asignaling pathway activation network analysis, the transcriptomesignature is used as input to a machine learning platform that outputsdrug classifications. The transcriptome signature is compared in thecomputer to a baseline transcriptome signature that represents a lesssenescent version of the patient's tissue or organ, and thetranscriptome signature is compared to a baseline transcriptomesignature that is constructed from more than one tissue or organtranscriptome signature. The computer may process the data to determinea specific determined drug of at least one or more of thesenoremediation drug, senolytic drug, antifibrotic drug, or preventativeaction drugs, and provide the determined drug as output. Then, thedetermined drug, one for each type, may be acquired and used in thetreatments described herein.

The method for treating senescence in a patient tissue or organ mayinclude applying an antifibrotic drug treatment protocol to the patientderived from a transcriptome signature of the patient, such as from thecomputer analysis thereof. The method may include wherein theantifibrotic drug treatment comprises at least one drug that destroysfibrotic cells. Alternatively, the method can include applying apro-fibrotic drug treatment, wherein the pro-fibrotic drug treatmentcomprises at least one pro-fibrotic drug that restores fibrotic cells.

In one embodiment, a method described herein, such as treatingsenescence in a patient tissue or organ may include (a) carrying out arescue step comprising applying a senoremediation drug treatmentprotocol to the patient; and (b) carrying out a removal step comprisingapplying a senolytic drug treatment protocol to the patient; wherein (a)and (b) may be done in any order, and at least one of the drugtreatments is derived from a transcriptome analysis of the patienttissue or organ. The method may also include (c) carrying out areplenishment step comprising introducing stem cells in a particulartissue or organ; and (d) carrying out a reinforcement step comprisingone or more methods to prevent further senescence or degradation of thetissue or organ.

In some aspects, the method can include repeating either (a) or (b) orboth at least once. In some aspect, the method can include (a) or (b)being carried out in either order. In some aspects, the method caninclude (c) carrying out a replenishment step comprising introducingstem cells in a particular tissue or organ. In some aspects, the methodcan include repeating (c) at least once. In some aspects, the method caninclude (c) being carried out in any sequence with respect to (a) and(b). In some aspects, the stem cells are mesenchymal or epithelial stemcells or both. In some aspects, the method can include (c) furthercomprising using a reinforcement drug treatment protocol to the patient.In some aspects, the method can include repeating (c) at least once. Insome aspects, the method can include (d) carrying out a reinforcementstep comprising one or more methods to prevent further senescence ordegradation of the tissue or organ. In some aspects, the method caninclude (d) being carried out in any sequence with respect to (a) (b)and (c). In some aspects, the method can include immunomodulation,cytoprotection, or stimulation of macrophages, or more than once. Insome aspects, the method can include (a) or (b) or both comprisingapplying an antifibrotic drug treatment protocol to the patient derivedfrom a transcriptome signature of the patient. In some aspects, themethod can include the antifibrotic drug treatment having at least onedrug that destroys fibrotic cells. In some aspects, antifibrotic drugtreatment comprises at least one drug that restores fibrotic cells. Insome aspects, the transcriptome signature is a signaling pathwayactivation network analysis. In some aspects, the transcriptomesignature is used as input to a machine learning platform that outputsdrug classifications. In some aspects, the transcriptome signature iscompared to a baseline transcriptome signature that represents a lesssenescent version of the patient's tissue or organ. In some aspects, thetranscriptome signature is compared to a baseline transcriptomesignature that is constructed from more than one tissue or organtranscriptome signature. In some aspects, the transcriptome signature isused as input to a deep neural network that outputs drugclassifications.

In some embodiments, a method of treating senescence in a patient tissueor organ can include: (a) carrying out a rescue step comprising applyinga senoremediation drug treatment protocol to the patient; and wherein atleast one of the drug treatments is derived from a transcriptomeanalysis of the patient tissue or organ. Here, step (a) can be performedalone. However, any of the other steps may also be performed as recitedherein.

The steps may be carried out in any order or repeated. The stem cellsmay be mesenchymal or epithelial stem cells or both. The method mayfurther include applying a reinforcement drug treatment protocol to thepatient, using, immunomodulation, cytoprotection, or stimulation ofmacrophages.

A transcriptome signature representing tissue or organ senescence may beused to develop at least one of the drugs used. The transcriptomesignature may be a signaling pathway activation network analysis, whichis performed on a computer with models as described herein. Thetranscriptome signature may be used in the following manner: as asignaling pathway activation network analysis, the transcriptomesignature is used as input to a machine learning platform that outputsdrug classifications. The transcriptome signature is compared to abaseline transcriptome signature that represents a less senescentversion of the patient's tissue or organ, and the transcriptomesignature is compared to a baseline transcriptome signature that isconstructed from more than one tissue or organ transcriptome signature.

The method may include applying an antifibrotic drug treatment protocolto the patient derived from a transcriptome signature of the patient.The method may include wherein the antifibrotic drug treatment comprisesat least one drug that destroys fibrotic cells or a pro-fibrotic drugtreatment comprises at least one drug that restores fibrotic cells.

The invention includes developing a personalized drug treatment, themethod may include: (a) receiving a first transcriptome signaturederived from a patient tissue or organ; (b) receiving a second firsttranscriptome signature derived from a baseline; (c) creating adifference matrix, such as in a computer with a model or neural networkor machine learning, using the profile of (a) and the profile of (b);(d) receiving a cellular signature library; (e) receiving a drugtherapeutic use library; (f) using the matrix of (c), the library of(d), and the library of (e) to provide input vectors to a machinelearning platform, wherein the machine learning platform outputsclassification vectors on one or more drugs, wherein the personalizeddrug treatment is comprised of the classification vectors.

The transcriptome signature may be based on a signature signalingpathway activation network analysis on a computer. One of thetranscriptome signatures is based an in silico signaling pathwayactivation network decomposition. One of the profiles may comprise aPearson correlation matrix. The personalized drug treatment may comprisea senescence treatment for the patient. The profile of (b)—the secondfirst transcriptome signature derived from a baseline—may be derivedfrom a non-senescent tissue or organ of the patient or another subject.The method may include the machine learning platform comprising one ormore deep neural networks. The method may include the machine learningplatform comprising at least two generative adversarial networks and maycomprise an adversarial autoencoder architecture. The personalized drugtreatment may be created by prescribing drugs identified by theclassification vectors at their lowest effective dose.

The invention includes a method of computationally, with a computer,designing a treatment protocol for a patient comprising one or moredrugs, the method comprising: (a) identifying a gene expressionsignature of the patient; (b) defining a patient score for signaturestaken from one or more patient tissues or organs; (c) selecting drugsbased upon (a) and/or (b); and (d) defining a lowest effectivecombination for each drug. The method may include the gene expressionsignature being based on a signature signaling pathway activationnetwork analysis, wherein gene expression signatures is based on an insilico signaling pathway activation network decomposition, wherein thegene expression signature comprises a transcriptome Pearson correlationmatrix. The method can then include one or more treatment steps with oneor more treatment drugs or treatment steps of any of the treatmentmethods described herein.

The protocol may be a senescence treatment for the patient. The methodmay include wherein: the signature of (a)—gene expression signature ofthe patient is derived, using a computer with appropriate algorithms ormodels (e.g., neural network) from a non-senescent tissue or organ ofthe patient or another subject, wherein (b) and (c) are carried out on amachine learning platform, wherein the machine learning platformcomprises at least two generative adversarial networks, wherein themachine learning platform comprises an adversarial autoencoderarchitecture, wherein the machine learning platform comprises one ormore deep neural networks.

In some embodiments, a computer program product can include anon-transitory computer readable medium having a computer readableprogram code embodied therein, the product being executable by aprocessor to perform a method for estimating the fractionalgluconeogenesis of a patient, the method comprising developing apersonalized drug treatment, comprising: (a) receiving a firsttranscriptome signature derived from a patient tissue or organ; (b)receiving a second first transcriptome signature derived from abaseline; (c) creating a difference matrix using the profile of (a) andthe profile of (b); (d) receiving a cellular signature library; (e)receiving a drug therapeutic use library; (f) using the matrix of (c),the library of (d) and/or (e), to provide input vectors to a machinelearning platform, wherein the machine learning platform outputsclassification vectors on one or more drugs, wherein the personalizeddrug treatment is comprised of the classification vectors.

In one aspect, at least one of the transcriptome signatures is based ona signature signaling pathway activation network analysis. In oneaspect, at least one of the transcriptome signatures is based an insilico signaling pathway activation network decomposition. In oneaspect, at least one of the profiles comprises a transcriptome Pearsoncorrelation matrix. In one aspect, the personalized drug treatmentcomprises a senescence treatment for the patient. In one aspect, theprofile of (b) is derived from a non-senescent tissue or organ of thepatient or another subject. In one aspect, the machine learning platformcomprises one or more deep neural networks. In one aspect, the machinelearning platform comprises at least two generative adversarialnetworks. In one aspect, the machine learning platform comprises anadversarial autoencoder architecture. In one aspect, the personalizeddrug treatment is created by prescribing drugs identified by theclassification vectors at their lowest effective dose.

The computer processing can include input and or processing of acomplete or partial schematic overview of the biochemistry ofsenescence. Additional information can be obtained in the incorporatedprovisional application regarding the biological pathways that can beuses as input and processing for determining a treatment, such asspecific drugs for the treatment. Accordingly, the biological pathwayscan be used in the methods described herein. Such biological pathwaysare described herein with some examples of computer processing thereoffor implanting the design of treatment protocols as recited herein.

A variety of cell-intrinsic and -extrinsic stresses that can activatethe cellular senescence program can be used as input for a simulation orother computer processing. The biological pathways that are known, suchas in the literature, can be analyzed for specific biological steps thatare performed. Modulation of the biological step either to increase theactivity or decrease the activity results in a cascading series ofevents in response to the modulated activity. The modulations can bewith drugs, substances, of other affirmative actions that effect amodulation of the biological pathway. This modulation can be measuredfor a defined biological step. The biological step and the change inresponse to the modulation activity can be used as inputs into computermodels, and such computer models can be trained on the data. Now, withthe increase in artificial intelligence and deep learning algorithms,such biological steps, the modulation activity, and the changed responsecan be used with such computer models for modeling biological pathways.This can allow for determining a modulation activity for one or morebiological steps. Such modulations activities can be real and based onthe simulations, such as being a real drug, substance, or medicalaction. The output of the computer models can be instructions or otherinformation for causing the modulation activity in order to obtain aspecific type of biological step modulation so that the end goal of aspecifically modulated biological pathway can be obtained. Accordingly,the biological pathways described herein, or in the incorporatedreferences and provisional applications, can be used as the biologicalpathways for the treatment protocols described herein.

In a specific example, the biological pathways can relate to senescence,and the modulation thereof.

The biological pathways related to senescence can be used for computermodels. Stressors are know to cause biological pathway modulation thatresults in senescence. For example, some stressors engage variouscellular signaling cascades and can ultimately activate p53, p16Ink4a,or both. Some stress types that activate p53 through DDR signaling canbe analyzed and computed. This can include computationally processingthe ROS to elicit the DDR by perturbing gene transcription and DNAreplication, as well as by shortening telomeres. The computer can alsocompute biological pathways of activated p53 that induces p21, whichinduces a temporal cell-cycle arrest by inhibiting cyclin E-Cdk2, whichcan be processed. The computer can also analyze how p16Ink4a alsoinhibits cell-cycle progression by targeting cyclin D-Cdk4 and cyclinD-Cdk6 complexes. Both p21 and p16Ink4a act by preventing theinactivation of Rb, thus resulting in continued repression of E2F targetgenes required for S-phase onset. Upon severe stress as modeled andcomputationally processed, temporally arrested cells that transitioninto a senescent growth arrest through a mechanism that is currentlyincompletely understood can be determined. Cells exposed to mild damagethat can be successfully repaired may resume normal cell-cycleprogression. On the other hand, cells exposed to moderate stress that ischronic in nature or that leaves permanent damage may resumeproliferation through reliance on stress support pathways, and suchinformation may be included in the data processing. This phenomenon(termed assisted cycling) is enabled by p53-mediated activation of p21,which can be taken into account when computationally determine atreatment, such as a drug treatment. Thus, the p53-p21 pathway caneither antagonize or synergize with p16Ink4a in senescence depending onthe type and level of stress that is used in the computationalprocessing. BRAF(V600E) is unusual in that it establishes senescencethrough a metabolic effector pathway. BRAF(V600E) activates PDH byinducing PDP2 and inhibiting PDK1 expression, promoting a shift fromglycolysis to oxidative phosphorylation that creates senescence-inducingredox stress, which can be taken into account in the computationalprocessing. Cells undergoing senescence induce an inflammatorytranscriptome regardless of the senescence inducing stress, and suchinflammatory transcriptome can be considered in determining thetreatment. Also, senescence-promoting and senescence-preventingactivities may be computed, and may be weighted relative to theirimportance. A senescence-reversing mechanism may be input or modeled orotherwise computed as part of the process.

A multi-step senescence model can also be input and computed. The modelcan be programmed to consider cellular senescence as a dynamic processdriven by epigenetic and genetic changes. An initial step computes theprogression from a transient to a stable cell-cycle arrest throughanalysis of a sustained activation of the p16Ink4a and/or p53-p21pathways. The model can consider the resulting early senescent cellsprogress to full senescence by downregulating lamin B1, therebytriggering extensive chromatin remodeling underlying the production of aSASP. The model can consider certain components of the SASP that arehighly conserved, whereas others may vary depending on cell type, natureof the senescence-inducing stressor, or cell-to-cell variability inchromatin remodeling. The computation can consider progression to deepor late senescence that may be driven by additional genetic andepigenetic changes, which can be computed, including chromatin budding,histone proteolysis and retrotransposition, driving furthertranscriptional change and SASP heterogeneity. The computation canconsider the efficiency with which immune cells dispose of senescentcells, and which may be dependent on the composition of the SASP. Theproinflammatory signature of the SASP can fade due to expression ofparticular microRNAs late into the senescence program, thereby perhapsallowing evasion of immuno-clearance, which can also be considered.

In some embodiments, a conceptual model can be computed in whichsenescent cells are subdivided into two main classes based on kineticsof senescence induction and functionality. The conceptual model canconsider that acute senescence is induced through cell-extrinsic stimulithat target a specific population of cells in the tissue. Acutesenescent cells self-organize their elimination through SASP componentsthat attract various types of immune cells. The conceptual model can beprogrammed to consider that induction of chronic senescence occurs afterperiods of progressive cellular stress or macromolecular damage whentarry cycling transitions into a stable cell-cycle arrest. Theconceptual model can consider that age-related immunodeficiency orproduction of less proinflammatory SASPs, immune cells may inefficientlyeliminate chronic senescent cells, allowing continuation of multi-stepsenescence. For example, the conceptual model may consider thatsenescence induced during cancer therapy may initially be acute andlater chronic in nature.

The computer models can be programmed and receive senescence input datafor computing how senescence promotes age-related tissue dysfunction.Senescence contributes to the overall decline in tissue regenerativepotential that occurs with ageing. The computer models can be programmedwith the observation that progenitor cell populations in both skeletalmuscle and fat tissue of BubR1 progeroid mice are highly prone tocellular senescence. Proteases chronically secreted by senescent cellsmay perturb tissue structure and organization by cleaving membrane-boundreceptors, signaling ligands, extracellular matrix proteins or othercomponents in the tissue microenvironment, which can affect thetreatment protocols described herein. In addition, other SASPcomponents, including IL-6 and IL-8, may stimulate tissue fibrosis incertain epithelial tissues by inducing EMT may be considered. Chronictissue inflammation, which is characterized by infiltration ofmacrophages and lymphocytes, fibrosis and cell death, is associated withageing and has a causal role in the development of various age-relateddiseases, which can be considered during identifying a treatment.

The matrix metalloproteinases and proinflammatory SASP components can bemodeled and considered in determining a treatment because of theirability create a tissue microenvironment that promotes survival,proliferation and dissemination of neoplastic cells. The model can beprocessed so that SASP can be modeled for increasing age-related tissuedeterioration through paracrine senescence, where senescent cells spreadthe senescence phenotype to healthy neighboring cells through secretionof IL-1b, TGFb and certain chemokine ligands. With gene expressionanalysis or pathway analysis it is possible to distinguish betweenpre-senescent and senescent cells signatures with the computations.

The models can be computed to consider that killing senescent cells canlead to rejuvenation of the tissue. For example, a modified FOXO4-p53interfering peptide can be considered that causes p53 and inducestargeted apoptosis of senescent cells (TASC), which neutralizes murineliver chemotoxicity from doxorubicin treatment. The TASC can beconsidered for restoring fitness, hair density, and renal function infast and naturally aged mice.

The model can be processed so that delaying senescence or even promotedeath of accumulating apoptosis-resistant senescent cells can be astrategy to prevent age related diseases. Tocotrienols (T3s) andquercetin (Q) can be input for modeling as senolytics agents (e.g.,small molecules that can selectively induce death of senescent cells).Both drugs are able to kill pre-senescent and senescent cells and can beused adjuvant therapy of cancer and preventive anti-aging strategies,and thereby can be used in the treatments herein.

The computational models can also consider fibrosis and senofibrosisconditions. The term fibrosis describes the development of fibrousconnective tissue as a reparative response to injury or damage, whichcan be considered during computing for treatment protocols. Fibrosis mayrefer to the connective tissue deposition that occurs as part of normalhealing or to the excess tissue deposition that occurs as a pathologicalprocess. The term senofibrosis describes the development of fibrousconnective tissue under influence of senescent cells, which can beconsidered during computing for treatment protocols. Senescent activatedcells lose their proliferative and collagen-producing capacity and haveincreased inflammatory property to produce inflammatory cytokinescompared with replicating activated “normal” cells. The computationalmodels can focus on two types of fibrosis and senofibrosis treatment:pulmonary (IPF) and liver.

The models can be processed to consider that fibrosis is a wound healingresponse that produces and deposits extracellular matrix (ECM) proteinsincluding collagen fibers, causing tissue scarring. Liver usuallyregenerates after liver injury. However, when liver injury andinflammation are persistent and progressive, liver cannot regeneratenormally and causes fibrosis. Hepatic stellate cells (HSCs) are theprimary source of activated myofibroblasts that produce extracellularmatrix in the liver. Progressive liver fibrosis results in cirrhosiswhere liver cells cannot function properly due to the formation offibrous scar and regenerative nodules and the decreased blood supply tothe liver. The model can perform such simulations. The model canconsider three main reasons for liver fibrosis: alcoholic fattydiseases; non-alcoholic fatty diseases; and viral hepatitis. In eachcase different mechanisms lead to fibrotic tissue formation, whichmechanisms can be processed to determine a suitable protocol.

The model can also consider that quiescent HSCs store VitaminA-containing lipid droplets, and HSCs lose lipid droplets when they areactivated. Transforming growth factor (TGF)-β and platelet-derivedgrowth factor (PDGF) are two major cytokines that contribute to HSCactivation and proliferation, resulting in activation intomyofibroblasts. Many other cytokines, intracellular signaling, andtranscription factors are involved in this process, and may beconsidered during computations.

The computational models can also consider activation and regression ofhepatic stellate cells. Quiescent hepatic stellate cells (HSCs) storeVitamin A containing lipid droplets and lose Vitamin A when the cellsare activated. Hepatic epithelial injury, such as death of hepatocytesand biliary epithelial cells, induces activation of HSCs directly orthrough cytokines released from immune cells including Kupffer cells,bone marrow-derived monocytes, Th17 cells, and innate lymphoid cells(ILC). Transforming growth factor-f (TGF-f), platelet-derived growthfactor (PDGF), interleukin-1f (IL-1f), IL-17, and intestine-derivedlipopolysaccharide (LPS) promote HSC activation. IL-33 promotes HSCactivation through ILC2. Autophagy in HSCs is associated with HSCactivation. The activated myofibroblast pool is mainly constituted byactivated HSCs, but biliary injury induces differentiation of portalfibroblasts to activated myofibroblasts. However, there is no evidenceof epithelial-mesenchymal transition for constituting the myofibroblastpool. After the cessation of causative liver injury, fibrosis startsregression, and activated HSCs induce apoptosis or revert into aquiescent state. Peroxisome proliferator-activated receptor 7 (PPAR7)expression in HSCs is associated with HSC reversal. Some activated HSCsbecome senescent, resulting in loss of profibrogenic property in whichp53 plays a role. Moreover, angiogenesis contributes to both fibrosisdevelopment and regression. As such, each may be considered whencomputing a therapeutic protocol.

The main pathways that are involved in modulation of hepaticinflammation can be categorized as (1) Upregulated and (2)Downregulated. The main pathways that are involved in formation ofcellular senescence in HSCs can be categorized as (1) Upregulated and(2) Downregulated. Both upregulation and downregulation of anybiological pathway, such as those described herein, may be consideredduring the computation of therapeutic protocols.

The main pathways which are involved in formation of cellular senescencephenotype in primary human hepatocytes (PHH). Data for the analysis istaken from LINCs transcriptomic dataset and computed as describedherein. Methanesulfonate is a DNA damage/senescence inducer, which maybe used in obtaining data to train the models. Liver senescence andliver fibrosis signatures hold the common features on the pathway level(analysis is based on the gene expression data using iPANDA, asdescribed further below.

The main pathways which are involved in formation of cellular senescencephenotype in primary human hepatocytes (PHH). Data for the analysis, andmodel computations for determining a therapeutic protocol can be takenfrom LINCs transcriptomic dataset. The following are Up-regulated: BRCA1Pathway Homologous Recombination Repair; JNK Pathway Insulin Signaling;Caspase Cascade Pathway Activated Tissue Trans-glutaminase; JNK PathwayGene Expression Apoptosis Inflammation Tumorigenesis Cell Migration viaSMAD4, STAT4, HSF1, TP53, MAP2, DCX, ATF2, NFATC3, SPIRE1, MAP1B, TCF15,ELK1, BCL2, JUN, PXN, and NFATC2; Caspase Cascade Pathway DNAFragmentation; TRAF Pathway Gene Expression via FOS and JUN; IF1AlphaPathway Gene Expression via JUN and CREB3; TNF Signaling PathwayApoptosis; PTEN Pathway Genomic Stability; VEGF Pathway Gene Expressionand Cell Proliferation via MAPK7; ErbB Family Pathway Gene Expressionvia JUN, FOS, and ELK1; PTEN Pathway Ca2+ Signaling; PTEN Pathway DNARepair; VEGF Pathway Prostaglandin Production; MAPK Family Pathway GeneExpression via ATF2, JUN, ELK1, NFKB2, and CREB3; HIF1Alpha Pathway; WNTPathway; ATM Pathway Cell Survival; and MAPK Family Pathway Translation.The following are Down-regulated: Ras Pathway Increased T-cell Adhesion;HGF Pathway Cell Adhesion and Cell Migration; IGF1R Signaling PathwayCell Migration; ILK Signaling Pathway Cell Migration Retraction; ILKSignaling Pathway Cell Cycle Proliferation; ILK Signaling Pathway G2Phase Arrest; ILK Signaling Pathway Cytoskeletal Adhesion Complexes; ILKSignaling Pathway Loss of Occludin Barrier Dysfunction; ATM Pathway CellCycle Checkpoint Control; Akt Signaling Pathway AR mediated apoptosis;Akt Signaling Pathway Apoptosis; Akt Signaling Pathway Cell CycleProgression; and Akt Signaling Pathway Elevation of Glucose Import. Therole of senescence of HSCs in liver fibrosis may be computed, andexperimental data using cell-specific genetic modifications to HSCs fromexperimental models of liver fibrosis in vivo can be used in thecomputation of treatment protocols.

There is no treatment for liver fibrosis still. The only way to avoid itis to prevent massive inflammation by rescuing or killing pre-senescentand senescent cells accordingly. Liver senescence and liver fibrosissignatures hold the common features on the pathway level (analysis isbased on the gene expression data using iPANDA package). The commonsignificant pathways involved into modulation liver fibrosis (andcirrhosis) are that can be considered in the computation models includethe following upregulated and down regulated pathways. Those upregulatedinclude: ILK Signaling Pathway Opsonization; ILK Signaling Pathway CellAdhesion; ILK Signaling Pathway Wound Healing; Akt Signaling Pathway ARmediated apoptosis; TRAF Pathway; IL-10 Pathway Stability Determination;EGF Pathway Rab5 Regulation Pathway; TRAF Pathway Gene Expression viaFOS and JUN; ILK Signaling Pathway Tumor Angiogenesis; Akt SignalingPathway NF-kB dependent transcription; HIF1Alpha Pathway Gene Expressionvia JUN and CREB3; Chemokine Pathway; STAT3 Pathway Growth Arrest andDifferentiation; TRAF Pathway Apoptosis; Erythropoietin Pathway GPIHidrolysis and Ca2+ influx; IL-10 Pathway; IL-10 Pathway InflammatoryCytokine Genes Expression via STAT3; ILK Signaling Pathway MMP2 MMP9Gene Expression Tissue Invasion via FOS; ErbB Family Pathway GeneExpression via JUN, FOS, and ELK1; Akt Signaling Pathway Regulation ofNa+ Transport; PAK Pathway Paxillin Disassembly; ILK Signaling PathwayCytoskeletal Adhesion Complexes; cAMP Pathway Glycogen Synthesis; andILK Signaling Pathway Cell Migration Retraction. Those downregulatedinclude: STAT3 Pathway Anti-Apoptosis; Akt Signaling Pathway Cell CycleProgression; Circadian Pathway; Growth Hormone Signaling Pathway ProteinSynthesis; and PTEN Pathway Migration.

The common significant pathways involved in formation of cellularsenescence and liver fibrosis that can be computed include those thatare upregulated and downregulated. Those upregulated include: ErbBFamily Pathway Gene Expression via JUN, FOS, and ELK1; HIF1Alpha PathwayGene Expression via JUN and CREB3; and TRAF Pathway Gene Expression viaFOS and JUN. Those downregulated include Akt Signaling Pathway CellCycle Progression. The common significant pathways involved intomodulation of IPF include those upregulated or downregulated. Thoseupregulated include: Cellular Apoptosis Pathway; KEGG Choline metabolismin cancer Main Pathway; KEGG Prostate cancer Main Pathway; NCI CXCR4mediated signaling events Main Pathway; NCI Syndecan 4 mediatedsignaling events Main Pathway; NCI TRAIL signaling Main Pathway; NCIValidated transcriptional targets of deltaNp63 isoforms Main Pathway;NCI Validated transcriptional targets of deltaNp63 isoforms Pathway(Pathway degradation of TP63); PTEN Pathway Adhesion or Migration; PTENPathway Angiogenesis and Tumorigenesis; PTEN Pathway Ca2+ Signaling;reactome Collagen biosynthesis and modifying enzymes Main Pathway; andreactome SMAD2, SMAD3, and SMAD4, heterotrimer regulates transcriptionMain Pathway. Those downregulated include: Growth Hormone SignalingPathway Gene Expression via SRF, ELK1, STAT5B, CEBPD, STAT1, STAT3; andreactome Tie2 Signaling Main Pathway.

The common significant pathways involved in formation of cellularsenescence in lung tissue can include those upregulated anddownregulated. Those upregulated include: Growth Hormone SignalingPathway Gene Expression via SRF, ELK1, STAT5B, CEBPD, STAT1, STAT3; KEGGCholine metabolism in cancer Main Pathway; KEGG Prostate cancer MainPathway; NCI CXCR4 mediated signaling events Main Pathway; NCI TRAILsignaling Main Pathway; PTEN Pathway Adhesion or Migration; PTEN PathwayAngiogenesis and Tumorigenesis; PTEN Pathway Ca2+ Signaling; reactomeCollagen biosynthesis and modifying enzymes Main Pathway; reactomeSMAD2, SMAD3, SMAD4 heterotrimer regulates transcription Main Pathway;and reactome Tie2 Signaling Main Pathway. Those downregulated include:Cellular Apoptosis Pathway; NCI Syndecan 4 mediated signaling eventsMain Pathway; NCI Validated transcriptional targets of deltaNp63isoforms Main; Pathway; NCI Validated transcriptional targets ofdeltaNp63 isoforms Pathway (Pathway degradation of TP63).

Cellular senescence can contribute to accelerating organ aging, and,among the pulmonary diseases that can be related to pulmonarysenescence, chronic obstructive pulmonary disease/emphysema (COPD) andidiopathic pulmonary fibrosis (IPF), are the most common and lethal.COPD and IPF are severe multifactorial pulmonary disorders characterizedby distinct clinical and pathologic features (“Global Strategy for theDiagnosis, Management, and Prevention of Chronic Obstructive PulmonaryDisease: GOLD Executive Summary Updated 2003” 2004; Noble et al. 2011).The date regarding clinical and pathological features can be used in thecomputational models that are processed for determining the therapeuticprotocols.

In all known types of cellular senescence, including replicativecellular senescence, stress-induced senescence, and oncogene-inducedsenescence, a permanent state of cell cycle arrest occurs that ismediated by the expression of p16INK4a and p21WAF1, 2 cell cycleinhibitors that are also well-recognized markers, to investigate thismechanism in vivo (Kim and Sharpless 2006; Campisi 2005; Mallette andFerbeyre 2007; Ohtani et al. 2004; Takeuchi et al. 2010). Alteredexpression of p16INK4a, p21WAF1, and b-galactosidase (a widely usedhistochemical marker of cellular senescence) have been demonstrated inIPF (Minagawa et al. 2010; Kuwano et al. 1996; Lomas et al. 2012). Thesemarkers are expressed strongly at sites of alveolar damage andhyperplasia, as well as in fibroblast foci localized in the discreteclusters of bronchiolar basal cells coexpressing the laminin-5-g2 chain(LAM5g2) and heat shock protein 27 (Hsp27) (Chilosi et al. 2006).According to review (Chilosi et al. 2013) several factors lead tosenescence in lungs, they are different for two types: idiopathicpulmonary fibrosis and chronic obstructive pulmonary disease/emphysemapathogenesis. This information may also be used in the computationalmodels for determining therapeutic protocols.

Methods for development of senescence drug treatments, that is, theselection of drugs, dosages, and cycles, are described herein. In thissection, we give an overview of the drug treatments, themselves, thatis, application of the personalized treatments once they have beendesigned, in a preferred embodiment, to the patient. In that patient, atissue or organ is identified to which the senescent treatment will beapplied.

In a preferred embodiment, one phase of the treatment involvessenoremediation, that is, a drug protocol of senoremediators, which aredrugs that restore or increase the amount of presenescent cells (cellsthat are typical or a young, healthy tissue or organ). Another phase ofthe treatment involves senolytic treatment, that is, a drug protocolthat involves restoring or that involves elimination or destruction ofsenescent cells in the tissue or organ of interest.

In another preferred embodiment, there is also an antifibrotic phase,that is, a drug protocol that addressing fibrotic cells in the tissue ororgan of interest. Antifibrotic may involve restoring senescent cells toa pre-senescent, non-fibrotic state, elimination or destruction offibrotic cells, or both.

Since such drug treatment protocols are highly specific, and based uponthe classification vectors of the analyses described herein, they maytake many forms. Methods in the art, such as Seim et. al., “Geneexpression signatures of human cell and tissue longevity”, npj Aging andMechanisms of Disease, 2, 16014 (2016), addresses transcriptomechanges/differences associated with senescence that are used to classifydrug protocols.

To examine gene expression strategies that support the lifespan ofdifferent cell types within the human body, one can obtain availableRNA-seq data sets and interrogated transcriptomes of various somaticcell types and tissues with reported cellular turnover, along with anestimate of lifespan, ranging from 2 days (monocytes) to effectively alifetime (neurons). Across different cell lineages, one can obtain agene expression signature of human cell and tissue turnover. Inparticular, turnover showed a negative correlation with theenergetically costly cell cycle and factors supporting genome stability,concomitant risk factors for aging-associated pathologies.

Comparative transcriptome studies of long-lived and short-lived mammals,and analyses that examined the longevity trait across a large group ofmammals (tissue-by-tissue surveys, focusing on brain, liver and kidney),have revealed candidate longevity-associated processes. Publiclyavailable transcriptome data sets (for example, RNA-seq) generated byconsortia, such as the Human Protein Atlas (HPA) can be used. They offeran opportunity to understand how gene expression programs are related tocellular turnover, as a proxy for cellular lifespan. Gene expressionpatterns are typically analyzed, in a preferred embodiment, usingPrincipal Component Analysis (PCA), as a first step.

The present invention involves examining aging transcriptome in whichthe transcribed genes in old to young people are compared to define aset first of genes which are more strongly expressed (activated) in oldpeople relative to young people and a second set of genes (repressed)which are less strongly expressed in old people relative to youngpeople. A preferred embodiment is herein described.

A rating approach can be used to rank the senescence treating propertiesof treatments first involves collecting the transcriptome datasets fromyoung and old patients and normalizing the data for each cell and tissuetype, evaluating the pathway activation strength (PAS) for eachindividual pathway and constructing the pathway cloud and screen fordrugs or combinations that minimize the signaling pathway clouddisturbance by acting on one or multiple elements of the pathway cloud.Drugs and combinations may be rated by their ability to return thesignaling pathway activation pattern closer to that of the youngertissue samples. The predictions may be then tested both in vitro and invivo on human cells and on model organisms such as rodents, nematodesand flies to validate the screening and rating algorithms.

In a preferred embodiment of the senescence treatment, a method forranking drugs, the method including; a. collecting young subjecttranscriptome data and old subject transcriptome data for one species toevaluate pathway activation strength (PAS) and down-regulation strengthfor a plurality of biological pathways; b. mapping the plurality ofbiological pathways for the activation strength and down-regulationstrength from old subject samples relative to young subject samples toform a pathway cloud map; and c. providing a rating for each of aplurality of drugs in accordance with a drug rating for minimizingsignaling pathway cloud disturbance (SPCD) in the pathway cloud map ofthe one species to provide a ranking of the drugs.

Pathway Activation and Pathway Activation Network Decomposition Analysis(iPANDA), is a preferred method of network analysis for the methodsdescribed herein.

Development of senescence treatments (in particular drug combinationsand protocols) as contemplated by the authors, are particularlycompatible with the signaling pathway activation network analysis asdescribed, for example, in U.S. 62/401,789 (Ozerov, filed September2016, now US 2018-0125865) and Ozerov et. al., “In silico PathwayActivation Network Decomposition Analysis (iPANDA) as a method forbiomarker development”, Nature Communications, 7: 13427, 2016, and bothincorporated by specific reference in their entity. Such methods includelarge-scale transcriptomic data analysis that involves insilico PathwayActivation Network Decomposition Analysis (iPANDA). The capabilities ofthis method apply to multiple data sets containing data on obtained, forexample, from Gene Expression Omnibus (GEO). Data sets in GEO areaccessed by identifier, or accession number, such as GSE5350.

Additionally, according to an embodiment of the present invention, thepathway cloud map shows at least one upregulated/activated pathway andat least one down-regulated pathway of the old subject relative to theyoung subject. Furthermore, according to an embodiment of the presentinvention, the pathway cloud map is based on a plurality of youngsubjects and a plurality of old subjects. Importantly, according to anembodiment of the present invention, the method is performed for anindividual to determine an optimized ranking of drugs for theindividual.

Further, according to an embodiment of the present invention, thesamples or biopsies are bodily samples selected from one or more of ablood sample, a urine sample, a biopsy, a hair sample, a nail sample, abreathe sample, a saliva sample, or a skin sample.

Yet further, according to an embodiment of the present invention, thepathway activation strength is calculated by dividing the expressionlevels for a gene n in the old subject samples by the gene expressionlevels of the young subject samples.

Additionally, according to an embodiment of the present invention, thepathway activation strength is calculated in accordance with

${SO} = \frac{\prod\limits_{i = 1}^{N}\; \lbrack{AGEL}\rbrack_{i}}{\prod\limits_{j = 1}^{N}\; \lbrack{RGEL}\rbrack_{J}}$

The [RGEL]i is an activator gene expression level and [RGEL]j is arepressor gene expression level) are expression level of activators genei and j, respectively.

Yet further, according to an embodiment of the present invention, todrugs or combinations that minimize the signaling pathway clouddisturbance (SPCD). Additionally, according to an embodiment of thepresent invention, the SPCD is a ratio of [AGEL]i, which is theactivator gene #i expression level, to [RGEL]j, which is the repressorgene #j expression level, and wherein this is calculated for activatorand repressor proteins in the pathway.

Cellular Network Analysis and iPANDA

There are well known method in the art (see, for example, U.S. Pat. No.8,623,592) for treating patients with methods for predicting responsesof cells to treatment with therapeutic agents. These methods involvemeasuring, in a sample of the cells, levels of one or more components ofa cellular network and then computing a Network Activation State (NAS)or a Network Inhibition State (NIS) for the cells using a computationalmodel of the cellular network. The response of the cells to treatment isthen predicted based on the NAS or NIS value that has been computed. Thepresent invention also comprises predictive methods for cellularresponsiveness in which computation of a NAS or NIS value for the cells(e.g., senescent cells) is combined with use of a statisticalclassification algorithm. A preferred method of iPANDA implementation isnow described. The method of transcriptomic data analysis, typicallyincludes receiving cell transcriptomic data of a control group (C) andcell transcriptomic data (S) of group under study for a gene,calculating a fold change ratio (fc) for the gene, repeating steps a andb for a plurality of genes, grouping co-expressed genes into modules,estimating gene importance factors based on a network topology, mappedfrom a plurality of the modules, in order to obtain an in silico PathwayActivation Network Decomposition Analysis (iPANDA) value, the iPANDAvalue having a Pearson coefficient greater than a Pearson coefficientassociated with another platform for manipulating the control celltranscriptomic data and the cell transcriptomic data of group understudy for the plurality of genes. Steps may also include determining abiological an in silico Pathway Activation Network DecompositionAnalysis (iPANDA) associated with at least one of the above the module,providing a classifier for treatment response prediction of a drug to adisease, wherein the disease is selected from a senescence and anotherdisease or disorder, applying at least one statistical filtering testand a statistical threshold test to the fc values, obtainingproliferative bodily samples and healthy bodily samples from patients,applying the drug to the patients, determining responder andnon-responder patients to the drug. The method also often includescomparing gene expression in at least one of selected signaling pathwaysand metabolic pathways, often associated with a drug.

One of the most relevant challenges in transcriptomic data analysis isthe inherent complexity of gene network interactions, which remains asignificant obstacle in building comprehensive predictive models.Moreover, high diversity of experimental platforms and inconsistency ofthe data coming from the various types of equipment—may also lead to theincorrect interpretation of the underlying biological processes.Although a number of data normalization approaches have been proposedover the recent years it remains difficult to achieve robust resultsover a group of independent data sets even when they are obtained fromthe same profiling platform. This may be explained by a range ofbiological factors, such as wide heterogeneity among individuals on thepopulation basis, variance in the cell cycle stage of the cells used ora set of technical factors, such as sample preparation or batchvariations in reagents.

A preferred embodiment of the present invention is compatible with thelarge-scale transcriptomic data analysis called in silico PathwayActivation Network Decomposition Analysis (iPANDA) as described herein.iPANDA is an effective tool for biologically relevant dimensionreduction in transcriptomic data.

Overview of a Preferred iPANDA Embodiment

Fold changes between the gene expression levels in the samples underinvestigation and an average expression level of samples within thenormal set is used as input data for the iPANDA algorithm. Since somegenes may have a stronger effect on the pathway activation than others,the gene importance factor has been introduced. Several approaches ofgene importance hierarchy calculation have been proposed during the lastfew decades. The vast majority of these approaches aim to enrichpathway-based models with specific gene markers most relevant for agiven study. While some of them use detailed kinetic models of severalparticular metabolic networks to derive importance factors, in others,gene importance is derived from the statistical analysis of the geneexpression data obtained for disease cases and healthy samples.

The iPANDA approach integrates different analytical concepts describedabove into a single network model as it simultaneously exploitsstatistical and topological weights for gene importance estimation. Thesmooth threshold based on the P values from a t-test performed on groupsof two contrasting tissue samples is applied to the gene expressionvalues. The smooth threshold is defined as a continuous function of Pvalue ranging from 0 to 1. The statistical weights for genes are alsoderived during this procedure. The topological weights for genes areobtained during the pathway map decomposition. The topological weight ofeach gene is proportional to the number of independent paths through thepathway gene network represented as a directed graph.

It is well known that multiple genes exhibit considerable correlationsin their expression levels. Most algorithms for pathway analysis treatgene expression levels as independent variables, which, despite thecommon belief, is not suitable when the topology based coefficients areapplied. Indeed, due to exchangeability, there is no dependence ofpathway activation values on how the topology weights are distributedover a set of coexpressed genes with correlated expression levels, andhence correlated fold changes. Thus, the computation of topologicalcoefficients for a set of coexpressed genes is inefficient, unless agroup of coexpressed genes is being considered as a single unit. Tocircumvent this challenge, gene modules reflecting the coexpression ofgenes are introduced in the iPANDA algorithm. The wide database of genecoexpression in human samples, COEXPRESdb, and the database of thedownstream genes controlled by various transcriptional factors areutilized for grouping genes into modules. In this way, the topologicalcoefficients are estimated for each gene module as a whole rather thanfor individual genes inside the module

The contribution of gene units (including gene modules and individualgenes) to pathway activation is computed as a product of their foldchanges in logarithmic scale, topological and statistical weights. Thenthe contributions are multiplied by a discrete coefficient which equalsto −1 or +1 in the case of pathway activation or suppression by theparticular unit, respectively. Finally, the activation scores, which werefer to as iPANDA values, are obtained as a linear combination of thescores calculated for gene units that contribute to the pathwayactivation/suppression. Therefore, the iPANDA values represent thesigned scores showing the intensity and direction of pathway activation.

Pathway Quality Metrics and iPANDA

Although currently there are several publicly available pipelines forbenchmarking the transcriptomic data analysis algorithms, our aim is togeneralize the approaches for pathway-based algorithm testing and revealthe common features of reliable pathway-based expression data analysis.We term these features “pathway analysis quality hallmarks”. Efficientmethods for pathway-based transcriptomic data analysis should be capableto perform a significant noise reduction in the input data and aggregateoutput data as a small number of highly informative features (pathwaymarkers).

Scalability (the ability to process pathways with small or large numbersof genes similarly) is another critical aspect that should be consideredwhen designing a reliable pathway analysis approach, since pathwayactivation values for pathways of different sizes should be equallycredible. The list of pathway markers identified should be relevant tothe specific phenotype or medical condition, and robust over multipledata sets related to the process or biological state underinvestigation. The calculation time should be reasonable to allowhigh-throughput screening of large transcriptomic data sets. To addressthe iPANDA algorithm in respect to these hallmarks and to fully assessits true potential and limitations, we have directly compared theresults obtained by iPANDA using the tissue and Microarray AnalysisQuality Control (MAQC)-I data sets with five other widely usedthird-party viable alternatives (GSEA8, SPIA9, Pathway Level Analysis ofGene Expression (PLAGE)26, single sample Gene Set Enrichment Analysis(ssGSEA) and Denoising Algorithm based on Relevant network Topology(DART)).

iPANDA as a Tool for Noise Reduction in Transcriptomic Data

One of the major issues that should be addressed when developing a noveltranscriptomic data analysis algorithm is the ability of the proposedmethod to reduce noise while retaining the biologically relevantinformation of the results. Since pathway-based analysis algorithms areconsidered dimension reduction techniques, the pathway activation scoresshould represent collective variables describing only biologicallysignificant changes in the gene expression profile.

In order to estimate the ability of the iPANDA algorithm to performnoise reduction while preserving biologically relevant features, weperformed an analysis of the well-known MAQC data set (GEO identifierGSE5350). It contains data for the same cell samples processed usingvarious transcriptome profiling platforms. A satisfactory pathway ornetwork analysis algorithm should reduce the noise level and demonstratea higher degree of similarity between the samples in comparison to thesimilarity calculated using gene set data.

To estimate gene level similarity only fold changes for differentiallyexpressed genes (t-test P value <0.05) were utilized. Pearsoncorrelation is chosen as a metric to measure the similarity betweensamples. Sample-wise correlation coefficients were obtained for the samesamples profiled on Affymetrix and Agilent platforms. Similar procedureis performed using pathway activation values (iPANDA values).

Notably, the similarity calculated using pathway activation valuesgenerated by the iPANDA algorithm significantly exceeds the onecalculated using fold changes for the differentially expressed genes(mean sample-wise correlation is over 0.88 and 0.79, respectively). Tofurther validate our algorithm, we directly compared its noise reductionefficacy with that of other routinely used methods fortranscriptome-based pathway analysis, such as SPIA, GSEA, ssGSEA, PLAGEand DART.

The mean sample-wise correlation between platforms is 0.88 for iPANDAcompared with 0.53 for GSEA, 0.84 for SPIA, 0.69 for ssGSEA, 0.67 forPLAGE and 0.41 for DART. Furthermore, the sample-wise correlationdistribution obtained using iPANDA values is narrowed to a range of 0.79to 0.94, compared with −0.08-0.80, 0.60-0.92, 0.61-0.74, 0.45-0.75 and−0.11-0.60 for GSEA, SPIA, ssGSEA, PLAGE and DART, respectively.

In a preferred embodiment, iPANDA does generally assign more weights togenes that tend to be reliably coexpressed using information fromCOEXPRESSdb database. The information from COEXPRESSdb is utilizedsolely for grouping genes into modules, and hence cannot introduce anyfavorable bias towards iPANDA in this assessment. Even when the featurefor grouping genes into modules is ‘switched off’, meaning that allgenes are considered individually and no information from COEXPRESSdb isbeing utilized, iPANDA scores show higher sample-wise similarity betweendata obtained using various profiling platforms compared with thesimilarity calculated on the gene level.

Biomarker Identification and Relevance and iPANDA

As a next step we address the iPANDA ability to identify potentialbiomarkers (or pathway markers) of the phenotype under investigation.One of the commonly used methods to assess the capability oftranscriptomic pathway markers to distinguish between two groups ofsamples (for example, resistance and sensitivity to treatment) is tomeasure their receiver operating characteristics area under curve (AUC)values. The capacity to generate a high number of biomarkers with highAUC values is a major requirement for any prospective transcriptomicdata analysis algorithm to be used in prediction models.

iPANDA Produces Highly Robust Set of Biomarkers

One of the most important shortcomings of modern pathway analysisapproaches is their inability to produce consistent results fordifferent data sets obtained independently for the same biological case.Here we show that iPANDA algorithm applied to the tissue data overcomesthis flaw and produces highly consistent set of pathway markers acrossthe data sets used in the study. The iPANDA algorithm is an advantageousmethod for biologically relevant pathway marker development comparedwith the other pathway-based approaches.

The common marker pathway (CMP) index is applied to drug treatmentresponse data for in order to estimate the robustness of the biomarkerlists. Pathway marker lists obtained for four independent data sets wereanalyzed. The calculation of pathway activation scores is performedusing the iPANDA algorithm and its versions with disabled gene groupingand/or topological weights. The ‘off’ state of topology coefficientsmeans that they are equal to 1 for all genes during the calculation.Also, the ‘off’ state for the gene grouping means that all the genes aretreated as individual genes. The application of the gene modules withouttopology-based coefficients reduces the robustness of the algorithm aswell as the overall number of common pathway markers between data sets.Turning on the topology-based coefficients just slightly increases therobustness of the algorithm. Whereas using topology and gene modulessimultaneously dramatically improves this parameter for both tissuetypes. This result implies that the combined implementation of the genemodules along with the topology-based coefficients serves as aneffective way of noise reduction in gene expression data and allows oneto obtain stable pathway activation scores for a set of independentdata.

PANDA biomarkers as classifiers for prediction models. High AUC valuesfor the pathway markers shown in suggest that iPANDA scores may beefficiently used as classifiers for biological condition predictionchallenges.

In order to classify the samples as responders or non-responders, therandom forest models were developed using iPANDA scores obtained fortraining sets of samples for each end point. Subsequently, performanceof these models is measured using validation sets. Matthew's CorrelationCoefficients (MCC), specificity and sensitivity metrics were applied toevaluate performance of the models. MCC metrics were chosen for the easeto calculate and due to their informativeness even when the distributionof the two classes is highly skewed. The similar random forest modelswere built using pathway activation (enrichment) scores obtained byother pathway analysis algorithms, including SPIA, GSEA, DART, ssGSEAand PLAGE. Moreover, to fully assess the performance of iPANDA-basedpaclitaxel sensitivity prediction models, we have trained the similarrandom forest models on four different gene expression subsets:expression levels of all genes (logGE), fold change for all genesbetween the training set and corresponding normals (logFC), expressionlevels of most differentially expressed genes (t-test P<0.05) (logDGE),and fold change in expression levels of most differentially expressedgenes (t-test P<0.05) between the training and corresponding normalbreast tissue data sets (logDFC). Logarithmic scale is used for trainingthe gene level models. All pathway-level and gene-level data is Z-scorenormalized separately for each GEO data set used.

Application of the pathway activation measurement implemented in iPANDAleads to significant noise reduction in the input data and henceenhances the ability to produce highly consistent sets of biologicallyrelevant biomarkers acquired on multiple transcriptomic data sets.Another advantage of the approach presented is the high speed of thecomputation. The gene grouping and topological weights are the mostdemanding parts of the algorithm from the perspective of computationalresources. Luckily, these steps can be precalculated only once beforethe actual calculations using transcriptomic data. The calculation timefor a single sample processing equals B1.4 s on the Intel (R) Corei3-3217U 1.8 GHz CPU (compared with 10 min for SPIA, 4 min for DART,about 10 s for ssGSEA, GSEA and PLAGE). Thus, iPANDA can be an efficienttool for high-throughput biomarker screening of large transcriptomicdata sets.

The use of merely microarray data for pathway activation analysis haswell-known limitations, as it cannot address individual variations inthe gene sequence and consequently in the activity of its product. Forexample, a gene can have a mutation that reduces activity of its productbut elevates its expression level through a negative feedback loop.Thus, the elevated expression of the gene does not necessarilycorresponds with the increase in the activity of its product.

Although the iPANDA algorithm is initially designed for microarray dataanalysis, it can also be easily applied to the data derived fromgenome-wide association studies (GWAS). In order to do so, GWAS data canbe converted to a form amenable for the iPANDA algorithm. Single-pointmutations are assigned to the genes based on their proximity to thereading frames. Then each single-point mutation is given a weightderived from a GWAS data statistical analysis40. Simultaneous use of theGWAS data along with microarray data may improve the predictions made bythe iPANDA method.

One of the rapidly emerging areas in biomedical data analysis is deeplearning. Recently several successful studies on microarray dataanalysis using various deep learning approaches on gene-level data havesurfaced. Using pathway activation scores may be an efficient way toreduce dimensionality of transcriptomic data for drug discoveryapplications while maintaining biological relevant features. From anexperimental point of view, gene regulatory networks are controlled viaactivation or inhibition of a specific set of signaling pathways. Thus,using the iPANDA signaling pathway activation scores as input for deeplearning methods could bring results closer to experimental settings andmake them more interpretable to bench biologists. One of the mostdifficult steps of multilayer perceptron training is the dimensionreduction and feature selection procedures, which aim to generate theappropriate input for further learning. Signaling pathway activationscoring using iPANDA will likely help reduce the dimensionality ofexpression data without losing biological relevance and may be used asan input to deep learning methods especially for drug discoveryapplications. Using iPANDA values as an input data is particularlyuseful for obtaining reproducible results when analyzing transcriptomicdata from multiple sources.

The gene expression data from different data sets is preprocessed usingGCRMA algorithm45 and summarized using updated chip definition filesfrom Brainarray repository (Version 18) for each data set independently.

Taken together, iPANDA demonstrates better performance for the noisereduction test in comparison to other pathway analysis approaches,suggesting its credibility as a powerful tool for noise reduction intranscriptomic data analysis. iPANDA ha strong ability to identifypotential biomarkers (or pathway markers) of the phenotype underinvestigation. One of the commonly used methods to assess the capabilityof transcriptomic pathway markers to distinguish between two groups ofsamples (for example, resistance and sensitivity to treatment) is tomeasure their receiver operating characteristics area under curve (AUC)values. The capacity to generate a high number of biomarkers with highAUC values is a major requirement for any prospective transcriptomicdata analysis algorithm to be used in prediction models.

There are several widely used collections of signaling pathwaysincluding Kyoto Encyclopedia of Genes and Genomes (KEGG), QIAGEN and NCIPathway Interaction Database. In this study, the collection of signalingpathways most strongly associated with various types of malignanttransformation in human cells were used, obtained from the SABiosciencescollection (sabiosciences.com/pathwaycentral.php). Using asenescence-specific pathway database can be used to ensure the presenceof multiple pathway markers for the particular condition underinvestigation. Each pathway contains an explicitly defined topologyrepresented as a directed graph. Each node corresponds to a gene or aset of genes while edges describe biochemical interactions between genesin nodes and/or their products. All interactions are classified asactivation or inhibition of downstream nodes. The pathway size rangesfrom about twenty to over six hundred genes in a single pathway.

The iPANDA approach for large-scale transcriptomic data analysisaccounts for the gene grouping into modules based on the precalculatedgene coexpression data. Each gene module represents a set of genes whichexperience significant coordination in their expression levels and/orare regulated by the same expression factors. Therefore the actualfunction for the calculation of the pathway p activation according tothe proposed iPANDA algorithm consists of two terms. While the first onecorresponds to the contribution of the individual genes, which are notmembers of any module, the second one takes into account thecontribution of the gene modules. Therefore the final function forobtaining a iPANDA value for the activation of pathway p, which consistsof the individual genes i and gene modules j, has the followinganalytical form:

${iPANDA}_{p} = {{\sum\limits_{i}G_{ip}} + {\sum\limits_{i}M_{ip}}}$

The contribution of the individual genes (Gip) and the gene modules(Mjp) is 15 computed as follows:

G_(ip) = w_(i)^(S) ⋅ w_(ip)^(T) ⋅ A_(ip) ⋅ lg (fc_(i))$M_{jp} - {{{\max \left( w_{i}^{S} \right)} \cdot \frac{1}{N}}{\sum\limits_{i}^{N}\left( {w_{ip}^{T} \cdot A_{ip} \cdot {\lg \left( {fc}_{i} \right)}} \right)}}$

Here fci is the fold change of the expression level for the gene i inthe sample 20 under study to the normal level (average in a controlgroup). As the expression levels are assumed to be logarithmicallynormally distributed and in order to convert the product over foldchange values to sum, logarithmic fold changes are utilized in the finalequation. Activation sign Aip is a discrete coefficient showing thedirection in which the particular gene affects the pathway given. Itequals +1 if the product of the 25 gene i has a positive contribution tothe pathway activation and −1 if it has a negative contribution. Thefactors wiS and wipT are the statistical and topological weights of the

${iPANDA}_{p} = {{\sum\limits_{i}G_{ip}} + {\sum\limits_{i}M_{ip}}}$G_(ip) = w_(i)^(S) ⋅ w_(ip)^(T) ⋅ A_(ip) ⋅ Ig(fc_(i))$M_{jp} = {{{\max \left( w_{i}^{S} \right)} \cdot \frac{1}{N}}{\sum\limits_{i}^{N}\left( {w_{ip}^{T} \cdot A_{ip} \cdot {\lg \left( {fc}_{i} \right)}} \right)}}$

with gene i ranging from 0 to 1. The derivation procedure for thesefactors is described in detail in the subsequent sections. Since lg(fci)and Aip values can be positive or negative, the iPANDA values for thepathways can also have different signs. Thus positive or negative iPANDAvalues correspond to pathway activation or inhibition respectively.

Obtaining Gene Importance Factors

In order to estimate the topological weight (wipT), all possible walksthrough the gene network are calculated on the directed graph associatedwith the pathway map. The nodes of the graph represent genes or genemodules, while the edges correspond to biochemical interactions. Thenodes which have zero incoming edges are chosen as the starting pointsof the walks and those which have zero outgoing edges are chosen as thefinal points. Loops are forbidden during walks computation. The numberof walks Nip through the pathway p which include gene i is calculatedfor each gene. Then wipT is obtained as the ratio of Nip to the maximumvalue of Njp over all genes in the pathway:

$w_{ip}^{T} = \frac{N_{ip}}{\max \left( N_{jp} \right)}$

The statistical weight depends on the p-values which are calculated fromgroup t-test for case and normal sets of samples for each gene. Themethod called p-20 value thresholding is commonly used to filter outspurious genes which demonstrate no significant differences betweensets. However, a major issue with the use of sharp threshold functionsis that it can introduce an instability in filtered genes and as aconsequence in pathway activation scores between the data sets.Additionally, the pathway activation values become sensitive to anarbitrary choice of the cutoff value. In order to address this issue,using a smooth threshold function is suggested. In the present study,the cosine function on logarithmic scale is utilized:

$w_{i}^{S} = \left\{ \begin{matrix}{0,} & {p > p_{\max}} \\{{\left( {{\cos \left( {\pi \frac{{\log \; p} - {\log \; p_{\min}}}{{\log \; p_{\max}} - {\log \; p_{\min}}}} \right)} + 1} \right)/2},} & {p_{\min} < p \leq I} \\{1,} & {p \leq p_{\min}}\end{matrix} \right.$

where pmin and pmax are the high and low threshold values. In this studyp-value thresholds equal to 10-7 and 10-1 respectively. For thethreshold values given over 58% of all genes pass high threshold andabout 12% also pass low threshold for the data under investigation.Hence over 45% of the genes in the data set receive intermediate wiSvalues. Therefore more stable results for pathway activation scoresbetween data sets can be achieved using this approach.

Grouping Genes into Modules

To obtain the gene modules, two independent sources of data wereutilized: 10 human database of coexpressed genes COEXPRESdb18 and thedatabase of the downstream genes controlled by human sequence-specifictranscription factors19. The latter is simply intersected with the genesfrom the pathway database used, while correlation data from COEXPRESdbis clustered using Euclidean distance matrix.

Distances were obtained according to the following equation:

r _(ij)=1−corr_(ij)

where corr_(ij) is correlation between expression levels of genes i andj. DBScan and hierarchical clustering with an average linkage criteriawere utilized to identify clusters. Only clusters with an averageinternal pairwise correlation higher than 0.3 were considered. Clustersobtained from the transcription factors database and coexpressiondatabase were recursively merged to remove duplicates. A pair ofclusters is combined into one during the merging procedure if theintersection level between clusters had been higher than 0.7. As aresult, a set of 169 gene modules which includes a total of 1021 uniquegenes is constructed.

Statistical Credibility of the iPANDA Values

The p-values for the iPANDA pathway activation scores are obtained usingweighted Fisher's combined probability test.

Algorithm Robustness Estimation

In order to quantitatively estimate the robustness of the algorithmbetween data sets, the Common Marker Pathway (CMP) index is introduced.The CMP 15 index is a function of the number of pathways considered asmarkers that are common between data sets. It also depends on thequality of the treatment response prediction when these pathways areused as classifiers. The CMP index is defined as follows:

${C\; M\; P} = {\frac{1}{n}{\sum\limits_{j = 1}^{n}{\sum\limits_{i}{{\ln \left( N_{i} \right)} \times \left( {{AUC}_{ij} - {AUC}_{R}} \right)}}}}$

where n is the number of data sets under study, Ni is the number ofgenes in the pathway i and AUCij is the value of the ROC area undercurve which shows the quality of the separation between responders andnon-responders to treatment when pathway i is used as classifier for thej-th data set. AUCR is the AUC value for a random classifier and equalsto 0.5. A pathway is considered as a marker if its AUC value is higherthan 0.8. The ln(Ni) term is included to increase the contribution ofthe larger pathways because they have a smaller probability to randomlyget a high AUC value. The higher values of the CMP index correspond tothe most robust prediction of pathway markers across the data sets underinvestigation, while zero value of CMP index corresponds to the emptyintersection of the pathway marker lists obtained for the different datasets.

Clustering of Data Samples

In order to apply iPANDA to the Paclitaxel treatment response predictionover a several independent data sets, the pathway activation values werenormalized to the Z-scores independently for each data set. The expectedvalues used for the Z-scoring procedure were adjusted to the number ofresponders and non-responders in the data set under study. The pairwisedistance matrix between samples utilized for further clustering isobtained using the

$D_{ij} = \sqrt{\frac{1}{N} \cdot {\overset{N}{\sum\limits_{p}}\left( {{iPANDA}_{ip} - {iPANDA}_{jp}} \right)^{2}}}$

Here Dij is the distance between samples i and j, N is the number of thepathway markers used for the distance calculation. iPANDAip and iPANDAipare the normalized iPANDA values for the pathway p for the samples i andj respectively. Normalization of iPANDA values to the Z-scores impliesthat all the considered pathway markers have an equal contribution tothe distance obtained. All distances were converted into similarities(1−Dij) before the clustering procedure. Hierarchical clustering usingWard linkage is performed on the distance matrix to divide the samplesinto groups.

Transcriptome (Gene Expression) Difference

In a preferred embodiment, two iPANDA transcriptome signatures, one froma senescent patient tissue or organ to be treated (or similar proxyprofile) and another representing a target, nonsenescent tissue ororgan, are compared to observe transcriptome (gene expression)differences. Principal component analysis is typically applied. Geneexpression trees, difference matrices matrix may also be use, as isknown in the art, for example using techniques know in the art. In apreferred embodiment, a difference matrix provides the vector inputs fora machine learning architecture as described below.

In a preferred embodiment, gene expression patterns are subjected toPrincipal Component Analysis (PCA). In an embodiment wherein manydifferent tissue samples are taken, rather than just two, severalclusters are formed, suggesting related biological functions for theseclusters. For example, the gastrointestinal tissues, esophagus, rectumand colon all grouped together, and hematopoietic tissues (bone marrowand spleen) and monocytes also clustered. Because transcriptomes offunctionally related cell types often exhibit substantial hierarchicalstructure a neighbor-joining gene expression tree can be generated basedon mean gene expression levels. Similar to the PCA results, bone marrowand spleen clustered with monocytes, while skeletal muscle and heartmuscle grouped together and were distinct from smooth muscle. Thus, forany given cell type, e.g., a neuron, epigenetic marks reflect both theprior (e.g., state in the germ layer and derived cell lineages) andpresent regulatory landscapes.

Differential Gene Expression of Cells and Tissues

In heart and skeletal muscle, 455 out of 12,044 genes are differentiallyexpressed (phylogenetic analysis of variance (ANOVA) P value≤0.01)compared with other cells and tissues. Approximately 44% of these geneswere associated with the tricarboxylic acid (TCA) cycle and respiration,in agreement with the metabolic organization and energy sources of thesetissues.

Neurons, which are critical for cognitive and motor functions, have celllifespans that likely exceed the lifespan of the organism. Comparingneurons to shorter-lived cells and tissues is conceptually similar tocomparing gene expression of long-lived mammals to related short-livedspecies, e.g., examining African mole rats against other rodents. 15Accordingly, neurons should possess a gene expression signatureassociated with low turnover/long lifespan, in addition to the patternsindicative of neuronal function. Out of 12,044 genes 1,438 weredifferentially expressed in neurons (P≤0.01) and gene set enrichmentanalysis showed enrichment for functions associated with lysosomes,proteasomes, ribosomal proteins and apoptosis. Neurons presented withreduced expression of 27 ribosomal proteins and multiple 20S proteasomesubunit genes, consistent with distinct protein metabolism required tofine-tune self-renewal and synaptic plasticity. This group of genes wasnot correlated with cell and tissue turnover, suggesting that thisexpression pattern is unique to long-lived neurons. Reduced proteinmetabolism, which may be induced by dietary restriction and otherinterventions, is known to associate with extended lifespan in a numberof model organisms. Furthermore, expression of the tumor suppressor p53(TP53) was significantly reduced (P≤0.001) in neurons, where it wasexpressed at a level gene expression pattern of cell and tissueturnover.

Inputs to Machine Learning Platform and iPANDA

In a preferred embodiment, a general design of the computationalprocedures that outputs drug classification of the invention are in foursequential steps: 1) transcriptomic similarity search, 2) protein targetbased search, 3) structural similarity based search, 4) transcriptomicsignature screening and 5) deep neural network based search.

Regarding (1) In silico Pathway Activation Network DecompositionAnalysis (iPANDA), can be applied to transcriptomic tissue-specificaging datasets obtained from Gene Expression Omnibus (GEO) with totalnumber of samples not less than 250 for each tissue. Tissue-specificcellular senescence pathway marker sets are identified. Only pathwaysconsiderably perturbed in senescent cells (pathways withiPANDA-generated p-values less than 0.05 are considered as pathwaymarkers). iPANDA scores are precalculated for Broad Institute LINCSProject data and were utilized for calculating transcriptomic compoundsimilarity. Euclidian or other similarity between vectors of iPANDAscores for senolytics and other compounds of interest are calculatedusing data on cell lines for corresponding tissue. Only previouslyidentified tissue-specific pathway markers were used for similaritycalculation.

Regarding 2) Using LINCS Project data on knockdown cell lines the sameprocedure are performed to identify key target genes involved in theaction of previously identified senolytic compounds D (Dasantinib), N(Navitoclax) and Q (Quercetin). The list of target genes is enriched byproteins likely to interact with these compounds using STITCH humandrug-target interaction database. Pharmacophore-based search andpublicly available docking algorithms are applied to identify thecompounds which specifically bind the identified targets with highestaffinity.

3) Structural similarity search is performed for three compounds alreadyknown to have senolytic properties (D,N,Q). Using publicly availablemolecular docking algorithms the importance weights for chemical groupswere defined. This information is utilized for QSAR-based structuregeneration and filtering. Compounds from pubchem database can also bescreened during the similar procedure in order to find structuralanalogues of D,N and Q.

4) To investigate potential effects of natural compounds without knownmolecular targets GEO and LINCS Project gene expression data are used.In both databases, datasets can be examined, consisting oftranscriptomes of cell lines before and after treatment with multipledifferent chemical compounds. For aging datasets scoring exactly thesame GEO datasets GSE66236, GSE69391, GSE18876, GSE21779, GSE38718,GSE59980, GSE52699, GSE48662 are used. It can be assumed that ananti-aging compound would affect an aged transcriptome to turn it into“younger” state. Mechanistically, this reflected a fact that if acertain regulatory pathway is increased (or decreased) with aging, itsend targets would increase (or decrease) expression with aging. Bysearching for compounds which decrease (or increase) the expression ofthose end targets, the drugs which target these aging-associatedpathways (some of its master regulators) could be discovered.

First, differentially expressed genes associated with aging are found,as well as differentially expressed genes after drug treatment. Formicroarray-based transcriptome data, a limma test of differential geneexpression is used. Each set of differentially expressed genes isordered accordingly to the following measure which takes into accountboth magnitude and statistical significance of the effect: FC max(0,−log(pvalue)), where PC is fold-change of gene expression between groupsand pvalue represents the result of limma test.

A statistically motivated score estimating anti-aging abilities of acompound is designed. A significantly up- or down-regulated gene weredefined as the ones with FDR<0.01 (after multiple-testing correction). AFisher exact test is performed which measured the association of twocharacteristics of each gene: being significantly downregulated afterthe drug treatment and being significantly upregulated during aging.

Vice versa, the same test is performed for significantly upregulatedgenes after the drug treatment versus significantly downregulated genesduring aging. The best of p-values of those two tests are taken as ascore for the given drug against aging. A multiple testing correction ofthe obtained p-values for the amount of compound under study can beperformed. The same methodology is applied for screening naturalcompounds within LINCS transcriptomic database that are similar to theeffects of other drugs, such as metformin.

5) The deep neural network based classifier of compound pharmacologicalclass can be trained on many compounds. Training data includedstructural data (QSAR, SMILES), transcriptomic response LINCS Projectdata on gene-level and pathway level (iPANDA) and drug-targetinteraction network from STITCH database. The specific class ofprospective senolytic compounds is declared during training. This classincluded compounds identified on the steps 1, 2, 3 of the study.

Established classifier accuracy is recorded after the class-balancing ofthe test 10 set. A list of senolytic compounds after scanning thedatabase of 300000+ compounds is obtained for further analysis. Topranking compounds are obtained on each of the steps and intersection isfound for each tissue independently. As a result compounds areidentified as having the best senolytic properties for the tissue. A setof structural analogues according to the procedure in step 3 isobtained, which possess similar molecular properties, and likelysenolytic properties.

6) Finding structural analogs of desired molecules. An aim also is tofind structural analogs of molecule of interest for protein-ligandinteraction. This approach is highly efficient for increasing thespecificity of binding with targets (proteins).

At the first step we provide an analysis of possible targets for thedrug compounds. This can be done in two ways: 1) using specific programsfor searching in databases for different interactions of molecules ofinterest with proteins/genes (e.g. STITCH); 2) article analysis of anexperimental data. In the case of a molecule chosen the second way as ithelps to select the best variants of experimentally approvedprotein-ligand interactions. From literature analysis n targets arechosen according to parameters: 1) specific binding of target withdrug(s); 2) the lowest IC50; 3) the presence of the structure in proteindata bank.

After that for all of the structures we applied docking for all possibleactive sites and additional pockets of binding. The best positions ofdrugs in target were chosen and after an additional docking is done withthe usage of algorithm of flexible chains.

Then all the structures of the target were analyzed according toalgorithm: 1) amount of hydrogen bonds 2) hydrophobic/hydrophilicinteractions 3) number n-n interactions. This information were usedfurther to understand the key principles by which molecule can bind intothe specific site of the target. According to such analysis one can findthe rules for a molecule to be modified in for better binding propertieswith specific target. With the usage of the software the analogs arefound according to the rule for the molecule. After that toxicology insilico test are provided with choosing non-toxic analogs. These newnon-toxic analogs were again docked into the binding site of the targetfor interactions analysis and those which showed the best score resultsare selected as most promising and perspective ones. Other structuralanalogs and conformers can be extracted from the Pubchem Database.

In a preferred embodiment, a deep neural network, similar to thatdescribed in, for example, Aliper et. al., “Deep learning applicationsfor predicting pharmacological properties of drugs and drug repurposingusing transcriptomic data”, Mol Pharm, 2016 Jul. 5; 13(7): 2524-2530,and Mamoshina et. al., “Applications of Deep Learning in Biomedicine”,Mol Pharm, 2016 Mar. 13(5), is used, in combination with a cellularsignature database such as the LINCS database and a drug therapeutic usedatabase such as MeSH, as inputs to the DNN in order to output drugclassifications to develop a therapeutic protocol, in this case tocategorize and choose drugs for a senescence or other treatmentprotocol. LINCS is the US Library of Network-Based Cellular SignaturesProgram aims to create a network-based understanding of biology bycataloging changes in gene expression and other cellular processes thatoccur when cells are exposed to a variety of perturbing agents. MeSH is(Medical Subject Headings) is the US National Library of Medicinecontrolled vocabulary thesaurus used for indexing articles for PubMed,the free search engine of references and abstracts on life sciences andbiomedical topics also from the US National Library of Medicine.

An AAE works by matching the aggregated posterior to the prior ensuresthat generating from any part of prior space results in meaningfulsamples. As a result, the decoder of the adversarial autoencoder learnsa deep generative model that maps the imposed prior to the datadistribution. An AAE can be used in applications such as semi-supervisedclassification, disentangling style and content of images, unsupervisedclustering, dimensionality reduction and data visualization. AAEs areused, for example, in generative modeling and semi-supervisedclassification tasks. Thus an AAE turns an autoencoder into a generativemodel. The AAE is often trained with dual objectives—a traditionalreconstruction error criterion, and an adversarial training criterionthat matches the aggregated posterior distribution of the latentrepresentation of the autoencoder to an arbitrary prior distribution.

In a preferred embodiment derived from (4) Kadurin, the method uses a7-layer AAE architecture with the latent middle layer serving as adiscriminator. As an input and output the AAE uses a vector of binaryfingerprints and concentration of the molecule. In the latent layer wealso introduced a neuron responsible for growth inhibition percentage,which when negative indicates the reduction in the number of tumor cellsafter the treatment. To train the AAE one uses a cell line assay datafor compounds profiled in a cell line. The output of the AAE can then beused to screen drug compounds, such as the 72 million compounds inPubChem, and then select candidate molecules with potentialanti-sensecent or properties.

The latest class of non-parametric approaches for deep generative modelsis known as generative adversarial network (GAN). In this new framework,initially proposed by Goodfellow, generative models are estimated via anadversarial process. In practice, two models are simultaneously trained:a generative model G that captures the data distribution, and adiscriminative model D that estimates the probability that a sample camefrom the training data rather than G. The training procedure for G is tomaximize the probability of D making an error. Thus, this framework doesnot correspond to the standard optimization problem as it is based on avalue function that one model seeks to maximize and the other seeks tominimize. The process terminates at a saddle point that is a minimumwith respect to one model's strategy and a maximum with respect to theother model's strategy. Because GANs do not require an explicitrepresentation of the likelihood, neither approximate inference norMarkov chains are necessary. Consequently, GANs provide an attractivealternative to maximum likelihood techniques.

Generative capabilities of deep adversarial network techniques open thedoors to new perspectives as it could contribute to overcome severallimitations of current data driven computational methods. For example,we can apply GANs on transcriptomics data for the generation of newsamples for a desired phenotypic groups and in chemoinformatics for theprediction of the physical, chemical, or biological properties andstructures of molecules. Quantitative structure-activity relationships(QSAR) and quantitative structure-property relationships (QSPR) arestill considered as the modern standard for predicting properties ofnovel molecules. To that end, many ML-based approaches have beendeveloped to tackle such problems, but recent results show that theDL-based methods match or outperform other state-of-the-art methods anddemonstrate better predictive performance, parsimony andinterpretability and web-based predictors are available on some cases.Furthermore, new methods based on convolutional neural networks are ableto perform predictions by directly using graphs of arbitrary size andshape as inputs rather than fixed feature vectors and one can expect tosee the development of more flexible deep generative architectures thatcan be applied directly to other structured data such as sequences,trees, graphs, and 3D structures. Thus, the deep adversarial networktechniques could be used to improve accuracy, generative capabilitiesand predictive power and address several issues including computationalcost, limited computation at each layer and limited informationpropagation across the graph.

Target prediction and mapping of bioactive small compounds and moleculesby analyzing binding affinities and chemical properties is another areaof research that makes extensive use of data-driven computationalmethods in order to optimize the use of data available in existingrepositories. Despite promising results and the availability ofweb-platforms to computationally identify new targets foruncharacterized molecules or secondary targets for known molecules suchas SwissTargetPrediction, in general, the available methods remain tooinaccurate for systematic binding predictions and physical experimentsremain the state of the art for binding determination. In this field,DL-based methods, such as the recently released methods AtomNet based ondeep convolutional neural networks have allowed to circumvent severallimitations and outperform more traditional computational methodsincluding RFs, SVMs for QSAR and ligand-based virtual screening. One canexpect that the development of DL-methods making use of the GANframework will also lead to significant improvement with respect toprediction accuracy and power.

In a preferred embodiment, the adversarial network and the autoencoderare trained jointly with SGD in two phases—the reconstruction phase andthe regularization phase—executed on each mini-batch. In thereconstruction phase, the autoencoder updates the encoder and thedecoder to minimize the reconstruction error of the inputs. In theregularization phase, the adversarial network first updates itsdiscriminative network to tell apart the true samples (generated usingthe prior) from the generated samples (the hidden codes computed by theautoencoder). The adversarial network then updates its generator (whichis also the encoder of the autoencoder) to confuse the discriminativenetwork. Once the training procedure is done, the decoder of theautoencoder will define a generative model that maps the imposed priorof p(z) to the data distribution.

In a preferred embodiment, the input layer is divided into a fingerprintpart and a concentration input neuron. In a preferred embodiment, an AAEis trained to encode and reconstruct not only molecular fingerprints,but also experimental concentrations. The Encoder consists of twoconsequent layers L1 and L2 with 128 and 64 neurons, respectively. Thedecoder consists of the two layers L′1 and L′2, comprising 64 and 128neurons respectively. The latent layer consists of 5 neurons, one ofwhich is the GI and the four others are discriminated with normaldistribution. Since we train an encoder net to predict ‘efficiency’against ‘senescence’ in a single neuron of latent layer, we divide thelatent vector in two parts—‘GI’ and ‘representation’. So we added aregression term to the encoder cost function. Furthermore, we restrictour encoder to map the same fingerprint to the same latent vectorindependently from input concentration by additional ‘manifold’ cost.Here we compute mean and variance of the concentrations through alldataset and then use them to sample concentrations for ‘manifold’ step.On each step we sample fingerprint from the training set and batch ofconcentration from normal distribution with given mean and variance. Thetraining net with ‘manifold’ loss is performed by maximization of cosinesimilarity between ‘representations’ of similar fingerprints withdifferent concentrations

All these changes resulted in a 5-step train iteration instead of a3-step in AAE basic model: (a) Discriminator trained to distinguishbetween given latent distribution and encoded ‘representation’, (b)Encoder trained to confuse Discriminator with generated‘representations’, (c) Encoder and Decoder trained jointly asAutoencoder; (d) Encoder trained to fit ‘score’ part of latent vector;(e) Encoder trained with ‘manifold’ cost.

The two first steps (a,b) are trained as usual adversarial networks. TheAutoencoder cost function is computed as a sum of logloss of fingerprintpart and mean squared error (MSE) of concentration parts and MSE is alsoused as a regression cost function. Example code for a preferred AAE isavailable at github.com/spoilt333/onco-aae.

Experimental/Simulations/Models

1. Single Biopsy (or Existing Individual Profile).

Single biopsy test of liver or lung is taken from the patient accordingto standard procedures in medical center as described in in thenhlbi.hih.gov website. For a lung biopsy, few samples of lung tissuefrom several places in lungs will be taken. The samples are examinedunder a microscope, transcriptome and gene expression profiles are alsoanalyzed. This procedure can help rule out other conditions, such assarcoidosis, cancer, or infection. Lung biopsy also can show how fardisease has advanced.

There are several procedures to get lung tissue samples.

Video-assisted thoracoscopy. This is the most common procedure used toget lung tissue samples. An endoscope is inserted with an attached lightand camera into chest through small cuts between ribs. The endoscopeprovides a video image of the lungs and allows to collect tissuesamples. This procedure must be done in a hospital.

Bronchoscopy. For a bronchoscopy, a thin, flexible tube through ispassed in nose or mouth, down a throat, and into airways. At the tube'stip are a light and mini-camera. They allow to see windpipe and airways.Then a forceps is inserted through the tube to collect tissue samples.

Bronchoalveolar lavage. During bronchoscopy, a small amount of saltwater (saline) is injected through the tube into lungs. This fluidwashes the lungs and helps bring up cells from the area around the airsacs. These cells are examined under a microscope.

Thoracotomy. For this procedure, a few small pieces of lung tissue areremoved through a cut in the chest wall between ribs. Thoracotomy isdone in a hospital.

For a liver biopsy, few samples of liver tissue from several places inliver will be taken. The samples are examined under a microscope,transcriptome and gene expression profiles are also analyzed.

There are several procedures to get live tissue samples.

Percutaneous Liver Biopsy. The health care provider either taps on theabdomen to locate the liver or uses one of the following imagingtechniques: ultrasound or computerized tomography (CT) and will takesamples with the needle.

Transvenous Liver Biopsy. When a person's blood clots slowly or theperson has ascites—a buildup of fluid in the abdomen—the health careprovider may perform a transvenous liver biopsy. A health care providerapplies local anesthetic to one side of the neck and makes a smallincision there, injects contrast medium into the sheath and take an xray. After this insert and remove the biopsy needle several times ifmultiple samples are needed.

Laparoscopic Liver Biopsy. Health care providers use this type of biopsyto obtain a tissue sample from a specific area or from multiple areas ofthe liver, or when the risk of spreading cancer or infection exists. Ahealth care provider may take a liver tissue sample during laparoscopicsurgery performed for other reasons, including liver surgery.

2. Pathway Signature Measurement

Transcriptomic Data:

From the GEO database (https://www.ncbi.nlm.nih.gov/geo/) data setscontaining gene expression data related to IPF patients and normalhealthy lung tissue used as a reference were downloaded (21 data sets).IPF and normal data from different data sets was preprocessed usingGCRMA algorithm and summarized using updated chip definition files fromBrainarray repository for each data set independently.

Differential genes were calculated using limma and deseq2 algorithms forgroups of comparison: IPF (IPF vs reference healthy lung tissue);Senescence (old vs reference young healthy lung tissue); Smoking(current smoker vs reference non-smoker); Age status data was availablefor 2 data sets and smoking status data was available for 1 data set.

Differential expression genes data was used as an input for iPANDAalgorithm in order to measure the pathway signature of each comparisongroup.

Pathway Database Overview:

There are several widely used collections of signaling pathwaysincluding Kyoto Encyclopedia of Genes and Genomes, QIAGEN and NCIPathway Interaction Database. In this study, we use the collection ofsignaling pathways most strongly associated with various types ofmalignant transformation in human cells obtained from the SABiosciencescollection (sabiosciences.com/pathwaycentral.php).

3. Compare Signature Profiles.

Signature profile for each comparison group can be constructed based oniPANDA (FIG. 2) p-values cut-off (p-value <=0.05) and common overlapamong different data sets: intersection cut-off threshold equal to 15was used for IPF data, 2 for senescence data and 1 for smoking data.

FIG. 2 shows the general scheme of an iPANDA calculation pipeline. Foldchanges between the gene-expression levels in the samples underinvestigation, and an average expression level of samples within thenormal set serves as input data for the iPANDA algorithm. The majorsteps of iPANDA algorithm include estimation of statistical weights (1),co-expression-based grouping of genes into modules (2), estimation oftopological weights (3) and calculation of iPANDA pathway activationscores (4).

4. Personalize the Treatment.

DNNs can be used as a tool to predict active compounds and generate acompounds with a desired efficacy. The application of DNN-based modelscan be used for personalization of compounds for individual patients andevaluation of the treatment efficacy and safety.

Machine learning approaches provide the tools of the analysis ofbiomedical data without prior assumption on the functional relations ofthis data. And Deep Neural Network (DNN) based approaches, such asmulti-layered feed forward neural networks, are able to fit the complexand sparse biomedical data and learn highly non-linear dependencies ofthe raw data without the modification of features of interest. And deeplearning is a state of the art method for many task from machine visionto language translation. But despite the fact, that biomedicine enteredthe era of “big data”, biomedical datasets are usually limited by samplesizes. And feature selection and dimensionality reduction of the featurespace usually increase the predictive power of the DNNs applied in thebiomedical domain (Aliper, Plis, et al. 2016).

A system can be provided that utilizes quantitative models with a deeparchitecture that is able to stratify compounds by their efficacy forthe individual patient based his or her personal profile. In part, thepersonal profile can include the biological pathways analyzed with thequantitative models. The following data could be used as input featureto the system: gene expression profiles and signaling pathway profiles,blood tests (Putin et al. 2016), protein expression profiles, clinicalhistory as well as a deep representation of the electronic health record(Miotto et al. 2016).

A system can be provided that utilizes the quantitative models with adeep architecture that is able to evaluate the efficacy of the proposedtreatment through the quantitative assessment of the health status ofthe patient, such a biological age, life expectancy, the probability ofsurvival. The following data could be used as input feature to thesystem: gene expression profiles and signaling pathway profiles, bloodtests, protein expression profiles, clinical history as well as a deeprepresentation of the electronic health record.

A system can be provided that utilizes the quantitative models with adeep architecture that is able to predict potential side effect of thetreatment. The following data could be used as input feature to thesystem: gene expression profiles and signaling pathway profiles, bloodtests, protein expression profiles, clinical history as well as a deeprepresentation of the electronic health record.

A system can be provided based on generative model with deeparchitecture (Kadurin et al. 2017) that is able to generate moleculeswith a desired properties, such as high efficacy, low toxicity, highbioavailability and the like. Generated molecules can be evaluated bythe DNN based systems through the efficacy and safety prediction.

Incubate Compound: Test in Laboratory for Effectiveness andIdentification, Design Therapy.

Treatment:

According to (Raghu et al. 2015) ATS/ERS/JRS/ALAT Clinical PracticeGuideline there are just a few agents recommended for IPF treatment:Nintedanib, a tyrosine kinase inhibitor with multiple targets,Conditional recommendation for use; Pirfenidone, inhibits TGF-βstimulated collagen production and reduces the production of fibrogenicmediators such as TGF-β, reduce production of inflammatory mediatorssuch as TNF-α and IL-1β in both cultured cells and isolated humanperipheral blood mononuclear cells, Conditional recommendation for use;Macitentan, bosentan, Dual endothelin receptor antagonists, Conditionalrecommendation against use; and Sildenafil, phosphodiesterase-5inhibitor, Conditional recommendation against use.

Some of the treatments are guided not to be used:_Anticoagulation(warfarin); Combination prednisone+azathioprine+N-acetylcysteine; orSelective endothelin receptor antagonist (ambrisentan).

The removal of the causative agent is the most effective intervention inthe treatment of liver fibrosis. This strategy has been shown effectivein most etiologies of chronic liver diseases. For patients withcirrhosis and clinical complications, liver transplantation is currentlythe only curative approach. Transplantation improves both survival andquality of life. However, in patients with HCV-induced cirrhosis, viralinfection recurs after transplantation, aggressive chronic hepatitisdevelops, and progression to cirrhosis is common.

According to (Cohen-Naftaly and Friedman 2011) there are several pointsof attack in developing antifibrotic agents that are described in detailin the following sections:_Eliminate the cause(s) of injury and theirmediators; Reduce inflammation and the immune response; Target specificsignaling: receptor-ligand interaction, intracellular signaling; Reducefibrogenesis, inhibit matrix synthesis; and Resolve fibrosis byincreasing scar matrix degradation, stimulating apoptosis of stellatecells, and BM or cell transplantation.

In each case the treatment is individual and obtained for every patientaccording to the etiology of the disease and patient condition (fibroticstage). More information about the strategies can be found at theaasld.org website.

The present invention can provide a strategy for treatment, which is notonly targeting the fibrotic conditions, but also pre-fibrotic andrelated/causing (e.g., cell senescence). Such approach allows a medicalpractitioner to increase chances for survival and decrease the speed ofdegradation processes or reverse of the organ.

Accordingly, a 5R strategy as described herein can be applied topatients with pre-senescent, senescent and fibrotic conditions. 5Rstrategy includes: Rescue; Remove; Replenish; reinforce; and Repeat,which are described as follows.

Stage 1. Rescue.

The first step of 5R strategy is rescuing pre-senescent cells in aparticular tissue (including liver and lungs). Pre-senescent phenotypeis considered potentially reversible.

In order to rescue the cells demonstrating pre-senescent phenotype thespecific set of possible interventions shall be applied. Theseinterventions include the treatment with the one senoremediator compoundor a combination of the senoremediator compounds from the list herein.Senoremediator compounds should be administered orally, by injection,sublingually, buccally, rectally, vaginally, cutaneously, transdermally,ocularly, oticly or nasally or any other way.

Stage 2. Remove.

This step is performed to eliminate the cells that already entered theirreversible senescent state. Senescent cells lose their function andpossess a constant danger to the surrounding cells as described above.Elimination of such cells may prevent surrounding cells to enter thesenescent phenotype by positive loop and restore the normal tissuefunctioning. In order to eliminate the cells demonstrating senescentphenotype the specific set of possible interventions shall be applied.These interventions include the treatment with the one senolyticcompound or a combination of the senolytic compounds from the listbelow. Senolytic compounds should be administered orally, by injection,sublingually, buccally, rectally, vaginally, cutaneously, transdermally,ocularly, oticly or nasally or any other way.

Stage 3. Replenish.

The second step leads to the general rejuvenation of the cells in thepopulation, but on the other hand, to the reduction in the total cellcount. This allows for the further replenish step to be used forrepopulation of the tissue with functional cells. Therefore the pool ofstem/progenitor cells in a particular tissue (including mesenchymal andepithelial stem cells in lungs, liver) should be activated in order toreplenish the tissue. The possible interventions needed to achieve thatgoal include the treatment with the one specific compound or acombination of the compounds from the list below. Importantly thecompounds should stimulate the proliferation of the stem cells, but onthe other hand prevent the unwanted effects related to the possibleuncontrolled proliferation and subsequent malignant transformation. Thecompounds should be administered orally, by injection, sublingually,buccally, rectally, vaginally, cutaneously, transdermally, ocularly,oticly or nasally or other method.

Stage 4. Reinforce.

This step is used to prevent the further potential degradation of thetissue (or organ). It may include the treatment with the one specificcompound or a combination of the compounds from the list below. Thesecompounds should demonstrate one of the following activities:immunomodulation in order to prevent possible malignant transformationand the accumulation of the senescent cells, cytoprotection in order toretain the functional state of the tissue, stimulation of themacrophages in order to achieve the specific state of senophagy (abilityto specifically engulf and digest senescent cells). The compounds shouldbe administered orally, by injection, sublingually, buccally, rectally,vaginally, cutaneously, transdermally, ocularly, oticly or nasally orother method.

Stage 5. Repeat.

The whole multi-stage longevity therapeutics pipeline (stages 1-4) canbe applied recurrently. The period between the therapies is definedindividually on the tissue (organ)-specific basis and may vary from 1month to 10 years.

In an embodiment, the first four steps Rescue; Remove; Replenish;Reinforce can be used as a multi-stage longevity therapeutics pipelineand can be applied more than once, and on an ongoing basis. The periodbetween the therapies is defined individually on a tissue, organ, andpatient specific basis and may vary from 1 month to 10 years betweentreatments, or may essentially be continually ongoing, for some or allof the steps.

EXAMPLES

The invention includes methods, system, drugs, apparatus, computerprogram product, among others, to carry out the following. It includes amethod of treating senescence in a patient tissue or organ.

Example 1

Assay Development and Validation for Testing Senolytic andSenoremediator Compounds in Skin and Lung.

The assay for senolytic and senoremediator compounds was developed basedon the present invention. A set of compounds for testing was selectedusing tissue-specific signatures of cellular senescence obtained withthe iPANDA algorithm. The compounds with previously measuredtranscriptional response profiles in one or more cell lines were scored(prioritized). Only significantly perturbed pathways from skin and lungcellular senescence signatures were used for scoring. For establishing alist of senolytic compound candidates the compounds similar toestablished senolytics (e.g., Navitoclax, Dasatinib) were selected. Forestablishing a list of senoremediator compound candidates the compoundsdemonstrating the reversed effect on the pathways under considerationcomparing to the senescence signatures in skin and lung were selected.

The assay development included the following steps: a) Selection of anappropriate senescence model for further compound validation; b)Selection of appropriate read-outs for compound testing; c) selectingcompounds showing significant response in senolytics assay; and d)selecting compounds showing significant response in senoremediationassay.

Step a) can include selection of an appropriate senescence model forfurther compound validation, such as: Replicative senescence. Somaticcells proliferate until they reach an irreversible growth arrest.Senescence occurs by simple subculturing of primary cells; Oxidativestress induced senescence. Cells undergo a transient stage of senescenceby chemical (e.g. H₂O₂); Irradiation-induced senescence. Cells undergo atransient stage of senescence by irradiation (e.g. UVA, UVB, IR);Drug-induced senescence. Low nanomolar concentration-treatments withmitomycin-C (MMC), a DNA inter- and intra-crosslinking agent; andParacrine senescence. Use conditioned medium from the drug-induced (MMC)senescence induction technique.

Primary human dermal fibroblasts and human lung fibroblasts (WI-38) wereselected as a model cell lines for the assay. Senescent cells differfrom their proliferative counterparts (e.g. morphology, β-galactosidaseactivity, oxidative stress, proliferation activity). The tests showedthat MMC treated cells and cells cultured in conditioned medium from theMMC-treated cells senescence applied can be used as a model ofsenescence for senolytic/senoremediation assay based on the set ofreadouts tested.

Step b) can include selection of appropriate read-outs for compoundtesting. The following read-outs were tested in MMC treated cells usingNavitoclax as senolytic positive control and nicotinamide mononucleotide(NMN) as senoremediation positive control (FIG. 3). FIG. 3 providesinformation for read-outs for senolytic and senoremediation compoundvalidation.

Step 3 c) can include selecting compounds that have shown significantresponse in senolytics assay, such as: Withaferin A, Argatroban,Flavopiridol, and Linifanib.

Step d) can include selecting compounds that have shown significantresponse in senoremediation assay, such as: Withaferin A, Lavendustin,and Sulforaphane.

The following is a description of the assay and validation process:

1. Cell Culture and MMC-Induced Senescence.

Human dermal fibroblasts (HDFs, CellnTec) were maintained in DMEM/F-12Ham (Sigma) supplemented with 10% FCS (Sigma) and 1% P/S (Sigma). Cellswere thawed and used at low passages (P3-P8).

On day −9, 1 Mio cells were thawed and allowed to adhere and grow. Thenon day −6 (FIG. 5) cells were seeded at a concentration of 5,000cells/cm² into two 96-well plates (1× caspase assay, 1× PrestoBlue).Remaining cells were further cultivated (mitotic cells control) andseeded the day before treatment (day 3) at a concentration of 35,000cells/cm² into 96-well plates. FIG. 5 shows the timeline of senolyticdrugs validation.

Mitomycin-C (MMC, Sigma) was dissolved in water (pH 7.02, sterilefiltered) and then further diluted in culture medium to a finalconcentration of 200 nM. On day −2 plated cells were treated with MMCfor two consecutive days, replacing the medium after 24 h with freshMMC-containing medium. After 48 h (day 0) MMC treatment was stopped bywashing with PBS and addition of fresh culture medium.

2. PrestoBlue Assay and Caspase-3/7 Assay.

On day 4 a total of four 96-well plates were ready for senolytic drugtesting (2× MMC-treated cells and 2× mitotic cells, each 1× PrestoBlueand 1× caspase-3/7 assay). Senolytic drugs were first tested at fiveconcentrations (10, 56, 316, 1778 and 10000 nM, n=3) and werere-evaluated at an optimized concentration range if drugs showedinteresting properties.

Metabolic activity of cells was evaluated before, directly after and twodays after the senolytic drug treatment. PrestoBlue (Invitrogen) wasdiluted in culture medium and fluorescence intensity was determined atEx./Em. 550/590 nm after 45 min.

After senolytic drug treatment cells were washed with HBSS+5 FCS (washsolution), followed by CellEvent Caspase-3/7 (Invitrogen) staining (6μM) at 37° C. for 1 h. The staining solution was removed and cell werefixed in formalin at room temperature for 15 min. Cellular DNA wasstained with DAPI (1 μg/mL) for 10 min in the dark. Then cells werewashed with wash solution and topped with a thin layer of wash solutionin order to keep them at 4° C. overnight.

Images were taken on an inverted epifluorescence microscope (Zeiss)equipped with DAPI and FITC filter cubes. At a magnification of 10×, 3images/well were taken (total of 9 images/drug and concentration).Acquired images were analyzed using ImageJ with the “Analyze particles”feature.

3. Exemplary Result for the Compound (INSSEN17)

FIG. 6A shows the PrestoBlue results of INSSEN17 treated cells for allfive tested concentration ranges prior (day 4) after (day 5) and twodays after the treatment (day 7). INSSEN17 did not affect the metabolicactivity of either, MMC-treated (blue bars) and mitotic cells (orangebars), up to a concentration of 6.49 μM. Slight cytotoxic effectsstarted to appear at 10 μM which dropped the metabolic activity ofMMC-treated and mitotic cells to 80-90% after the treatment. WhileMMC-treated cells stagnated in metabolic activity on day 7, mitoticcells dropped by another 30% in comparison to the DMSO controls. FIG. 6Ashows the PrestoBlue results of INSSEN17. Statistic: Student's t-test(two-sided, unpaired, unequal variances): ** p<=0.01, *** p<=0.001.Error bars are 95% confidence intervals of the mean.

Caspase-3/7 assay (FIG. 6B) showed a favorable concentration-dependentincrease in caspase-positive MMC-treated cells up to a concentration of6.49 μM while significantly differing from the mitotic cells. At thesame time mitotic cells did not differ from the mitotic DMSO control.The increase in caspase-positive MMC-treated cells could not besupported by the PrestoBlue assay.

INSSEN17 exhibited a moderate to good senolytic activity in comparisonto the Navitoclax positive control (2.5% vs. 4% caspase-positive cells).Even though PrestoBlue assay did not support this message, it suggestedthat there are no toxic effects concerning the overall metabolicactivity of the cells up to a concentration of 6.49 μM.

Looking at the current results, INSSEN17 comes close to the positivecontrol and should definitely be considered as a possible senolytic drugfor further evaluations.

Example 2

Analysis with iPanda of Single Biopsy (or Existing Individual Profile)of the Tissues.

Four pathways are disregulated in opposite directions among IPF andsenescence groups: two pathways related to hormone signaling (via SRF,ELK1, STAT5B, STAT1, STAT3 and CEBPD) and Tie2 signaling are upregulatedin senescence data and downregulated in IPF data; four pathways(Cellular Apoptosis Pathway, NCI Validated transcriptional targets ofdeltaNp63 isoforms (Main Pathway, pathway degradation of TP63)) aredownregulated in senescence data compared to IPF. Syndecan-4 mediatedsignaling tend to be upregulated in IPF data compared to senescencedata. CXCR4 signaling, PTEN signaling (adhesion or migration, Ca2+signaling, angiogenesis and tumorigenesis branches), SMAD2-SMAD3-SMAD4transcriptional regulation signaling and collagen biosynthesis andcholine metabolism seem to be upregulated in both groups of comparison._Four pathways related to syndecan-1 mediated cell adhesion, invasionand migration signaling seem to be the similarly upregulated in both IPFand smoking groups while 5 signaling pathways related to a particularbranch of hormone signaling (via SRF, ELK1, STAT5B, STAT1, STAT3 andCEBPD), sphingolipid metabolism, Spry regulation of FGF signaling,type-1 hemidesmosome assembly seem to be disregulated in oppositedirections between two groups.

IPF and smoking comparison overlap data is presented as shown in FIG. 4.Four pathways related to syndecan-1 mediated cell adhesion, invasion andmigration signaling seem to be the similarly upregulated in both IPF andsmoking groups while 5 signaling pathways related to a particular branchof hormone signaling (via SRF, ELK1, STAT5B, STAT1, STAT3 and CEBPD),sphingolipid metabolism, Spry regulation of FGF signaling, type-1hemidesmosome assembly seem to be disregulated in opposite directionsbetween two groups.

Senescence and smoking comparison overlap data is presented as shown inFIG. 4. Data has nine one-way directed signaling pathways amongsenescence and smoking groups: seven pathways are upregulated and twopathways are downregulated. 21 signaling pathways are disregulated inopposite directions among senescence and smoking groups: 11 pathways aredownregulated in senescence data while are upregulated in smoking group;10 pathways are upregulated in senescence data while are downregulatedin smoking data. Four signaling pathways seem to have indefinite statusamong two groups. FIG. 4. represents Venn diagram of iPANDA signaturepathways for each group of comparison. FIG. 4 shows a Venn diagram ofiPANDA signature pathways for each group of comparison. Two commonpathways seem to be relevant for three groups of comparisons. Growthhormone signaling pathway (via SRF, ELK1, STAT5B, STAT1, STAT3 andCEBPD) is upregulated in smoking and senescence data compared todownregulation in IPF data. Another pathway is Tie2 signaling:downregulated in both IPF and smoking groups and upregulated insenescence data.

The aging clock algorithm can be used to compute a personalizedtreatment protocol. Biological clocks are compatible with and useful forproviding guidance to aging and senescent assessment and remediation.Thus, they are compatible with and useful with the current invention.Such biological aging assessment is typically a first step inaging/longevity treatment and remediation. The figures in theprovisional illustrate some exampled biological aging clocks of that areuseful with the current invention. In addition, while discussion of themachine learning and deep learning methods below is discussed in thecontext of biological aging assessment, many of the methodologies aresimilar to and are adapted in this invention for development of thesenescent therapies, drug and otherwise, disclosed.

FIG. 7 illustrates a transcriptomic clock method for biological agingassessment, compatible with the current invention. The correlationbetween actual chronological age (x-axis) with predicted age (y-axis)for healthy individuals using the validation set. The red linerepresents the linear regression decision boundary line. Values for r,R2 and p-value are provided at the top of the figure. Note that the termDisease0 in this and other figures simply means healthy/control subjectswere used for such biological aging assessment.

FIG. 8 illustrates the delta (difference between assigned (predicted)biological age and actual chronological age) bar plots grouped by ageranges for healthy people based on an exemplary validation set asdescribed. The delta here is defined as the difference between assignedbiological age and real age. Thus, for example, a positive value meansthat a particular organism or person has been assigned a higher age thanactual chronological age.

FIG. 9 illustrates distribution on number of samples by age for healthyindividuals in the validation set. Blue (darker) and green (lighter)values are actual chronological age and assigned biological ages,respectively. For relatively healthy people, not surprisingly, assignedbiological is close to chronological age. During the training of the GAfor the selection of the most important age-related genes, differentderivative-based methods were applied for the initialization of the GA.

FIG. 10 illustrates an example epsilon-prediction accuracy for healthyindividuals. The accuracy in function of the Epsilon is shown on theFIG. 10. The epsilon-prediction accuracy is defined as follows:

${:{ɛ - {prediction}}} = \frac{\sum\limits_{i = 1}^{N}{1_{A}\left( f_{i} \right)}}{N}$

Where f_(i) is the predicted value, 1_(A) is an indicator A∈[y_(i)−ε;y_(i)+ε]

For example, if epsilon=0 and yi=45, the DNN correctly recognizes thissample if the prediction of the sample belongs to the interval.

FIG. 11 is a histogram illustrates clustering using t-SNE clusteringalgorithm by age for healthy individuals. Color bar indicates the age ofthe sample. For this particular example, there are no clearly definedclusters of healthy individuals by age.

Table A provides 49 genes that are determined to be significantlyimportant, in a preferred embodiment, for age prediction grouped bydisease and molecular function category.

TABLE A Category List of genes in each category Metabolism and energyACACB, SCD, ALDOC, SMOX, homeostasis AMACR, HTRA1, ARG1, HLCS, HSD3B7,PECI Hypertension and hypoxia PTGDS, HPGD, NT5E, TMSB4Y, ADORA2B, ACTN1,SNTB2. Neuropathy NETO2, GRM2, CACNA1I, NRCAM, CCT5, BAIAP2, QPRT,TMEM18, PPP1R9B, Genomic stability TOP1MT, PARP3, NOTCH1, TAF7, TINF2,CHTOP, CTBP1, CBX7, RRP1, RNF144, PNPT1, C16orf42 Smooth muscleconstruction ADORA2B, SOD1 Age-related macular HTRA1 degeneration Tumorangiogenesis CD248, VASH1, SERTAD3, TNFSF8, YWHAE, CRK, CBLL1, CDCA7L,E2F4 Inflammation AKIRIN2, DEFB123, PLXNC1, PSMD12; RELA

Table B lists of 100 gene names and abbreviations, all human, used fortranscriptome clock analysis in a preferred embodiment.

TABLE B Gene Name Ensembl gene ID David Gene Name Species ACACBENSG00000076555 acetyl-CoA carboxylase beta(ACACB) Homo sapiens ADORA2BENSG00000170425 adenosine A2b receptor(ADORA2B) Homo sapiens AKIRIN2ENSG00000135334 akirin 2(AKIRIN2) Homo sapiens AMACR ENSG00000242110alpha-methyl acyl-CoA racemase(AMACR) Homo sapiens ANKRD54ENSG00000100124 ankyrin repeat domain 54(ANKRD54) Homo sapiens ARFGAP3ENSG00000242247 ADP ribosylation factor GTPase Homo sapiens activatingprotein 3(ARFGAP3) ARHGAP26 ENSG00000145819 Rho GTPase activatingprotein Homo sapiens 26(ARHGAP26) BAIAP2 ENSG00000175866 BAI1 associatedprotein 2(BAIAP2) Homo sapiens BET1 ENSG00000105829 Bet1 golgi vesicularmembrane Homo sapiens trafficking protein(BET1) BPNT1 ENSG000001628133′(2′), 5′-bisphosphate nucleotidase 1(BPNT1) Homo sapiens C16orf42ENSG00000007520 TSR3, Acp Transferase Ribosome Homo sapiens MaturationFactor Cl7orf48 ENSG00000170222 ADP-Ribose/CDP-Alcohol Diphosphatase,Homo sapiens Manganese C1orf77 ENSG00000160679 Chromatin Target Of PRMT1Homo sapiens C9orf91 ENSG00000157693 Transmembrane Protein 268 Homosapiens CACNA1I ENSG00000100346 calcium voltage-gated channel subunitalpha1 Homo sapiens I(CACNA1I) CBLL1 ENSG00000105879 Cbl proto-oncogenelike 1(CBLL1) Homo sapiens CBX7 ENSG00000100307 chromobox 7(CBX7) Homosapiens CCT5 ENSG00000150753 chaperonin containing TCP1 subunit 5(CCT5)Homo sapiens CD248 ENSG00000174807 CD248 molecule(CD248) Homo sapiensCDCA7L ENSG00000164649 cell division cycle associated 7 Homo sapienslike(CDCA7L) CDK6 ENSG00000105810 cyclin dependent kinase 6(CDK6) Homosapiens CLDN14 ENSG00000159261 claudin 14(CLDN14) Homo sapiens CLIC3ENSG00000169583 chloride intracellular channel 3(CLIC3) Homo sapiensCOBRA1 ENSG00000188986 Negative Elongation Factor Complex Homo sapiensMember B CRK ENSG00000167193 CRK proto-oncogene, adaptor protein(CRK)Homo sapiens CTBP1 ENSG00000159692 C-terminal binding protein 1(CTBP1)Homo sapiens DAPP1 ENSG00000070190 dual adaptor of phosphotyrosine and3- Homo sapiens phosphoinositides 1(DAPP1) DBNDD2 ENSG00000244274dysbindin domain containing 2(DBNDD2) Homo sapiens DEFB123ENSG00000180424 defensin beta 123(DEFB123) Homo sapiens DERPCENSG00000168802 Chromosome Transmission Fidelity Factor 8 Homo sapiensDHTKD1 ENSG00000181192 dehydrogenase E1 and transketolase domain Homosapiens containing 1(DHTKD1) E2F4 ENSG00000205250 E2F transcriptionfactor 4(E2F4) Homo sapiens FANCL ENSG00000115392 Fanconi anemiacomplementation group Homo sapiens L(FANCL) FLJ10374 ENSG00000105248coiled-coil domain containing 94 Homo sapiens FLJ43093 ENSG00000255587RAB44, Member RAS Oncogene Family Homo sapiens FZD1 ENSG00000157240frizzled class receptor 1(FZD1) Homo sapiens GALNS ENSG00000141012galactosamine (N-acetyl)-6- Homo sapiens sulfatase(GALNS) GALNT6ENSG00000139629 polypeptide N- Homo sapiensacetylgalactosaminyltransferase 6(GALNT6) GATAD2A ENSG00000167491 GATAzinc finger domain containing Homo sapiens 2A(GATAD2A) GLT1D1ENSG00000151948 glycosyltransferase 1 domain containing Homo sapiens1(GLT1D1) GPA33 ENSG00000143167 glycoprotein A33(GPA33) Homo sapiensGRM2 ENSG00000164082 glutamate metabotropic receptor 2(GRM2) Homosapiens HSD3B7 ENSG00000099377 hydroxy-delta-5-steroid dehydrogenase, 3Homo sapiens beta- and steroid delta-isomerase 7(HSD3B7) LDOC1LENSG00000188636 leucine zipper down-regulated in cancer 1 Homo sapienslike(LDOC1L) LIPN ENSG00000204020 lipase family member N(LIPN) Homosapiens LMCD1 ENSG00000071282 LIM and cysteine rich domains 1(LMCD1)Homo sapiens LOC100130298 ENSG00000258130 hCG1816373-like(LOC100130298)Homo sapiens LOC285908 ENSG00000179406 Long Intergenic Non-ProteinCoding RNA Homo sapiens 174 LOC613038 ENSG00000258130 SAGA complexassociated factor 29 Homo sapiens pseudogene(LOC613038) LOC643905ENSG00000221961 Proline Rich 21 Homo sapiens LOC652784 NA NA Homosapiens LOC653884 NA serine/arginine-rich splicing factor 10-like Homosapiens LOC729338 ENSG00000224786 Centrin 4, Pseudogene (CETN4P) Homosapiens LOC731444 NA NA Homo sapiens LRP3 ENSG00000130881 LDL receptorrelated protein 3(LRP3) Homo sapiens MFNG ENSG00000100060 MFNGO-fucosylpeptide 3-beta-N- Homo sapiens acetylglucosaminyltransferase(MFNG) NETO2 ENSG00000171208 neuropilin and tolloid like 2(NETO2) Homosapiens NRCAM ENSG00000091129 neuronal cell adhesion molecule(NRCAM)Homo sapiens NTSR2 ENSG00000169006 neurotensin receptor 2(NTSR2) Homosapiens NUDT5 ENSG00000165609 nudix hydrolase 5(NUDT5) Homo sapiensPACSIN2 ENSG00000100266 protein kinase C and casein kinase substrateHomo sapiens in neurons 2(PACSIN2) PARP3 ENSG00000041880poly(ADP-ribose) polymerase family Homo sapiens member 3(PARP3) PARP8ENSG00000151883 poly(ADP-ribose) polymerase family Homo sapiens member8(PARP8) PECI ENSG00000198721 Enoyl-CoA Delta Isomerase 2 Homo sapiensPLXNC1 ENSG00000136040 plexin C1(PLXNC1) Homo sapiens PNPT1ENSG00000138035 polyribonucleotide nucleotidyltransferase Homo sapiens1(PNPT1) PPP1R9B ENSG00000108819 protein phosphatase 1 regulatorysubunit Homo sapiens 9B(PPP1R9B) PSMD12 ENSG00000197170 proteasome 26Ssubunit, non-ATPase Homo sapiens 12(PSMD12) QPRT ENSG00000103485quinolinate phosphoribosyltransferase(QPRT) Homo sapiens RAB3DENSG00000105514 RAB3D, member RAS oncogene Homo sapiens family(RAB3D)RELA ENSG00000173039 RELA proto-oncogene, NF-kB Homo sapienssubunit(RELA) RGMB ENSG00000174136 repulsive guidance molecule familymember Homo sapiens b(RGMB) RNASET2 ENSG00000026297 ribonucleaseT2(RNASET2) Homo sapiens RNF144 ENSG00000151692 Ring Finger Protein 144AHomo sapiens RRP1 ENSG00000160214 ribosomal RNA processing 1(RRP1) Homosapiens S100A9 ENSG00000163220 S100 calcium binding protein A9(S100A9)Homo sapiens SERTAD3 ENSG00000167565 SERTA domain containing 3(SERTAD3)Homo sapiens SGPL1 ENSG00000166224 sphingosine-1-phosphate lyase1(SGPL1) Homo sapiens SIGLEC7 ENSG00000168995 sialic acid binding Iglike lectin 7(SIGLEC7) Homo sapiens SLC25A19 ENSG00000125454 solutecarrier family 25 member Homo sapiens 19(SLC25A19) SLC38A10ENSG00000157637 solute carrier family 38 member Homo sapiens10(SLC38A10) SOD1 ENSG00000142168 superoxide dismutase 1, soluble(SOD1)Homo sapiens SRPRB ENSG00000144867 SRP receptor beta subunit(SRPRB) Homosapiens TAF7 ENSG00000178913 TATA-box binding protein associated factorHomo sapiens 7(TAF7) TCTN3 ENSG00000119977 tectonic family member3(TCTN3) Homo sapiens TIGD7 ENSG00000140993 tigger transposable elementderived Homo sapiens 7(TIGD7) TINF2 ENSG00000092330 TERF1 interactingnuclear factor 2(TINF2) Homo sapiens TMEM18 ENSG00000151353transmembrane protein 18(TMEM18) Homo sapiens TMSB4Y ENSG00000154620thymosin beta 4, Y-linked(TMSB4Y) Homo sapiens TNFSF8 ENSG00000106952tumor necrosis factor superfamily member Homo sapiens 8(TNFSF8) TRIM7ENSG00000146054 tripartite motif containing 7(TRIM7) Homo sapiensTSPAN10 ENSG00000182612 tetraspanin 10(TSPAN10) Homo sapiens VKORC1L1ENSG00000196715 vitamin K epoxide reductase complex Homo sapiens subunit1 like 1(VKORC1L1) VTI1B ENSG00000100568 vesicle transport throughinteraction with Homo sapiens t-SNAREs 1B(VTI1B) YWHAE ENSG00000108953tyrosine 3-monooxygenase/tryptophan 5- Homo sapiens monooxygenaseactivation protein epsilon(YWHAE) ZNF259 ENSG00000109917 ZPR1 ZincFinger Homo sapiens ZNF544 ENSG00000198131 zinc finger protein544(ZNF544) Homo sapiens ZNF583 ENSG00000198440 zinc finger protein583(ZNF583) Homo sapiens ZNF697 ENSG00000143067 zinc finger protein697(ZNF697) Homo sapiens ZNF763 ENSG00000197054 zinc finger protein763(ZNF763) Homo sapiens

Under the pressure of environmental factors and hereditarycharacteristics, the rate of aging naturally varies between individuals.As a result, biological age as defined by biomarkers often differsbetween individuals of the same chronological age. Biomarkers ofbiological aging again are the objective physiological indicators oftissues and organ conditions that are used to assess personal agingrates. Aging is of course associated with health risks, inability tomaintain homeostasis and eventual death prognosis of age-relateddiseases.

The biomarkers of biological aging as described herein can evaluate theeffectiveness of anti-aging remedies. This is of importance aspopulations in developed nations throughout the world are rapidly aging,and the search and identification of efficient anti-aging interventions,has never been more essential.

Because aging is a complex multifactorial process with no single causeor treatment (Zhavoronkov 2011; Trindade, 2013) that affects most if notall tissues and organs of the body, the currently available biomarkersin the art do not accurately represent the health state of the entireorganism or individual systems, and do not provide accurate and usefulmeasures of biological age. Furthermore, several of them are not easilymeasured. Thus biomarkers based on not only quantifiable but also easilymeasurable characteristics are still required.

Usually, identifying and developing biomarkers is a multi-steps processthat includes proof of concept, experimental validation and analyticalperformance validation. Nevertheless, alternative approaches based on insilico methods can also be used in order to improve and speed up thedevelopment and validation process of these biomarkers. The use of moreeffective computational approaches for the development of biomarker isfavored by two technological trends. First of all, the accumulation ofhigh-throughput data generated from different research areas such asproteomics, genomics, chemoproteomics and phenomics. The secondtechnological trend is the progress made in computational sciences that,combined with increasingly powerful computational resources, allows thedevelopment of repurposing algorithms but also of software's forretrospective analysis as well as the maintenance of web-based databaseswhich are required for the gathering and classification of theexperimental data (Lavecchia, 2016). Using these computationalresources, various techniques such as Machine Learning (ML) areroutinely used in biomarker development.

Although Deep Learning (DL) methods were initially developed for dealingwith task such as pattern, voice and image recognition (Oquab 2014),they can also be used to improve the efficiency of in silico techniquesapplied for biomarkers identification. DL-based methods are indeed ableto overcome many current limitation of more traditional in silicotechniques. For instance, for integrating biomedical data which arecomplex. The modern DL techniques include powerful approaches with deeparchitecture, called Deep Neural Networks (DNNs). Neural Networks arecollections of neurons (also called units) connected in an acyclicgraph. Neural Network models are often organized into distinct layers ofneurons.

For most neural networks, the most common layer type is thefully-connected layer in which neurons between two adjacent layers arefully pairwise connected, but neurons within a single layer share noconnections. One of the main features of DNN is that neurons arecontrolled by non-linear activation functions. This non-linearitycombined with the deep architecture make possible more complexcombinations of the input features leading ultimately to a widerunderstanding of the relationships between them and as a result to amore reliable final output. DNNs have already been applied for manytypes of data ranging from structural data to chemical descriptors ortranscriptomics data (Mayr 2016, Wang 2014, Ma 2015). Because of thisflexibility and adaptability of DNN for learning from large range ofdata, DNNs are now considered as an interesting computational approachfor tackling many current biomedical related issues (Mamoshina 2016, Xu2015, Hughes 2015).

Recently, Putin et al. (Putin, 2016) have published promising resultsdemonstrating the capacity of DNN-based methods to accurately predictbiological age and identify a set of the most relevant biomarkers fortracking physiological processes related to aging. In their study, thefeatures, a set of 41 biomarkers for each sample, used as inputs for theDNN were extracted from tens of thousands of blood biochemistry samplesfrom patients undergoing routine physical examinations. Although beinghighly variable in nature, blood biochemistry test is in practice verysimple to perform and it is approved for clinical use and as aconsequence, commonly used by Physicians. An effective DNN structure wasobtained using 56177 samples for the training phase (fitting ofhyperparameters) with the remaining 6242 samples used for validation.The interesting results obtained for predicting biological age show thatDNN-based approach outperform many traditional machine learning methodsincluding GBM (Gradient Boosting Machine), RF (Random Forests), DT(Decision Trees), LR (Linear Regression), kNN (k-Nearest Neighbors),ElasticNet, SVM (Support Vector Machines).

Furthermore, PFI (Permutation Features Importance) method was used tocompute the relative importance of each biomarker used to estimatebiological age. This information can be used in two ways. Firstly, aseach biomarker aims at measuring a specific biological mechanism, thisranking can be exploited to optimize anti-aging strategies by targetingthe most critical biological processes identified as playing a key rolein the onset and propagation of aging. Secondly, this list can be usedto reduce the number of initial inputs required to generate accurateprediction of biological age. Regarding this second point, the resultspresented in the study show that although each sample initially containsup to 46 biomarkers, the performance of DNNs remained remarkably stablewith an input comprising only the 10 first markers with the highest PFIscore. Thus, PFI provide a ranked list of biomarkers that can be used toselect the most robust and reliable features for predicting age.

The growing body of evidence on experimental data on life extension ofmodel organisms suggests the feasibility of finding interventionspromoting human longevity (Moskalev A 2017). However, the restrictedexperimental possibilities of studying human aging and overall lowtranslation rate from model organisms to the human clinic in othertherapeutic areas (Mak, Evaniew, and Ghert 2014) complicates the searchof desirable anti-aging therapies and only a few geroprotectors,anti-aging molecules, shown potential efficacy in humans (A. Aliper etal. 2016; I. Thomas and Gregg 2017; A. M. Aliper et al. 2015).

For the past several decades, research in understanding the molecularbasis of human aging has progressed significantly. Changes in geneexpression are associated with numerous biological processes, cellularresponses and disease states most likely play the crucial role in agingprocess. (de Magalhães, Curado, and Church 2009).

Because biological aging is not a single signature, but highly specificin terms or organs, tissues, systems, and other granular aspects of theorganism (including humans), an effective and useful biological clockmust utilize many biomarkers from many tissues and organs. The followingare some preferred examples.

Energy Metabolism:

Glycolysis, glucose oxidation, fatty acids oxidation are main sources ofATP generation, which is crucial for the viability of tissue withhigh-energy demand, such as muscle tissue, and especiallycardiomyocytes. Aging process triggers abnormalities in metabolism andenergy homeostasis (Ma and Li 2015), and aging biomarkers specific toenergy metabolism are a subject of the present invention.

Hypertension and Hypoxia:

Prostaglandins are critical to regulate vasodilation andvasoconstriction and to maintain vascular homeostasis. Balance ofvasodilating and vasoconstricting agents is important to maintain normalvascular function. Aging process shift the balance toward apro-constrictive agents and hypertension, which is the common vascularcomplication in elderly (Pinto 2007).

No matter the particular biomarkers being assessed by a biological agingassessment compatible with the current invention, a preferred embodimentof the deep learning computational approach for both the currentinvention and biological aging assessment is as follows. Firstly, aspecific type of DNN called Deep Feature Selection (DFS) is trained onblood gene expression samples using standard backpropagation algorithm.Secondly, the DFS model is applied to select a set of age-related genesusing different DNN-based feature selection methods combined into oneensemble model via genetic algorithm.

During the first step, DFS model is trained, for example, on 4000healthy human blood gene expression samples extracted from GEO(GSE33828). DFS (Li et al.) is type of neural network with severalspecific characteristics. Firstly, DFS adds a particularly hidden layer,called a weighted layer, which bridges one to one input features withneurons in the weighted layer. After that the neurons in the weightedlayer are connected one to many with neurons in first normal hiddenlayer of deep feed forward multilayer neural network. Secondly, DFSintroduces several regularization terms in the neural network lossfunction. An exemplary final loss function expression is as follows:

${{\min\limits_{\theta}\mspace{11mu} {f(\theta)}} = {{l(\theta)} + {\lambda_{1}\left( {{\frac{1 - \lambda_{2}}{2}{w}_{2}^{2}} + {\lambda_{2}{w}_{1}}} \right)} + {\alpha_{1}\left( {{\frac{1 - \alpha_{2}}{2}{\sum\limits_{k = 1}^{K + 1}{W^{(k)}}_{F}^{2}}} + {\alpha_{2}{\sum\limits_{k = 1}^{K + 1}{W^{(k)}}_{1}}}} \right)}}},$

where l(θ) is the log-likelihood of data, λ1, λ2, a1 and a2 areregularization terms. K is the number of hidden layers.

w

₂ ² and

w

₁ stand for the l2 and l1 norm for weight in weighted layer,respectively.

*

_(F) stands for the Frobenius norm and

*

₁ for the matrix norm. The last two terms are the ElasticNet-based termsthat control smoothness/sparsity for weights of weighted layer. Theyreduce the model complexity and speed up the training. After DFS modelwas trained the absolute values of the weights in the weighted layercould be used as ranking list for the input features (genes).

During the second step, DNN-based feature selection methods are used toselect age-related genes. Each method produces a ranked list of relativeimportance for each gene. In addition to the ranking of input featuresavailable with the DFS model itself, other methods have been applied.This includes the permutation feature importance (PFI) method aspreviously described in [Putin et al.], the heuristic variable selection(HVS) (Yacoub et al.) and methods based on output derivatives. Thenotable characteristic of these methods is that they can be applied toalready trained DNNs. It is not necessary to iteratively retrain DNNs asrequired by the forward or backward feature selection methods.

Heuristic Variable Selection (Yacoub et al.) is a zero first ordermethod designed for measuring the relative importance of input featuresof neural network. The method requires that the set of weight values andinformation related to the DNN structure as inputs. In a preferredembodiment, the relative importance of each given input feature iscomputed as follows:

$S_{i} = {\sum\limits_{j \in H}\left( {\frac{w_{ji}}{\sum\limits_{i^{\prime} \in I}{w_{{ji}^{\prime}}}}{\sum\limits_{k \in O}\frac{w_{kj}}{\sum\limits_{j^{\prime} \in H}{w_{{kj}^{\prime}}}}}} \right)}$

where I, H, O are the number of input, hidden and output layers,respectively. Note wji denotes the weight between neurons j and i. Afterthe training of the DNN and the computation of S for each input featurei, the set of S values can be assembled as a ranked list.

There are various of first order methods to measure the relativeimportance of an input feature. These methods used either the derivativeof the error or the output of the neural network with respect to thisinput feature to establish the ranked list. An interesting property ofthe derivative-based methods is that they can be applied to any type ofdifferentiable h are specific to each derivative-based method. Theprocedure to compute the average relevance of the input feature and howthe derivative term is included. Here we consider the long-studiedderivative-based methods described in details in (Dorizzi et al.), (Rucket al.), (Refenes et al.), (Czernichow et al.). In the followingformulas,

$\frac{{{df}\mspace{14mu} {j\left( x^{l} \right)}}\mspace{14mu}}{{dx}_{i}}$

means an output derivative of unit j of the network with respect to xiin xl point, Fj(xl) in is an output of the network with ul as input, Nis the number of samples. If specified, M is a number of outputs of thenetwork, var stands for the variance, q₉₅ or 95% percentile. In thetable below the relative importance Si of an input feature i ispresented by methods.

The biological aging assessment uses, as an example:

1) The model developed by Ruck et al., which is the following:

$S_{i} = {\sum\limits_{l = 1}^{N}{\sum\limits_{j = 1}^{g}{{\frac{\partial f_{j}}{\partial x_{i}}\left( x^{l} \right)}}}}$

(2) Refenes et al., have developed three different models:

$S_{i} = {\frac{1}{N}\frac{{var}\left( x_{i} \right)}{{var}\left( {{f(x)} - y} \right)}{\sum\limits_{l}\left( {\frac{\partial f}{\partial x_{i}}\left( x^{l} \right)} \right)^{2}}}$$S_{i} = {\frac{1}{N^{1/2}}\frac{\left( {\sum\limits_{l}\left( {{\frac{\partial f}{\partial x_{i}}\left( x^{l} \right)} - {\sum\limits_{j}{\frac{\partial f}{\partial x_{i}}\left( x^{j} \right)}}} \right)^{2}} \right)^{1/2}}{\sum\limits_{l}{\frac{\partial f}{\partial x_{i}}\left( x^{j} \right)}}}$$S_{i} = {\frac{1}{N}{\sum\limits_{l}{{\frac{\partial f}{\partial x_{i}}{\left( x^{l} \right) \cdot \frac{x_{i}}{f\left( x^{l} \right)}}}}}}$

3) The model of Dorizzi et al. takes the following form:

$S_{i} = {q_{95}\left( {{\frac{\partial f}{\partial x_{i}}(x)}} \right)}$

4) The model of Czernichow et al. is as follows:

$S_{i} = \frac{\sum\limits_{i = 1}^{N^{\prime}}\left( {\frac{\partial f}{\partial x_{i}}\left( x^{l} \right)} \right)^{2}}{\max_{j}\left( {\sum\limits_{i = 1}^{N^{\prime}}\left( {\frac{\partial f}{\partial{xj}}\left( x^{l} \right)} \right)^{2}} \right)}$

The final list of ranked genes is obtained by combining the differentlists described above using simple genetic algorithm (GA). In apreferred embodiment, the GA proceeds according to the following.

The initial population of genes is initialized by all feature rankinglists obtained by applying the aforementioned feature selectionalgorithms on both DNN and DFS models.

On each iteration the GA performed 35 crossover operations between itspopulations and 15 mutation operations, during which random genes wereinjected in the training of GA. Thus at each iteration, 50 DNNs weretrained. Convergence of the GA was reached after 50 epochs and finalgene ranking list was obtained. The best DNN model in the GA got 0.79 ofcoefficient of determination and 4.2 mean absolute error on validationdataset. On FIG. 7, one can see the performance of the DNN forpredicting the age of healthy individuals (#=0.79).

Cellular Life Span, Aging, Tissue-Specific Age Prediction, Thus,Biological Aging Assessment Compatible with the Current Invention.

As discussed above, different cell and tissues exhibit differentexpression patterns, different aging patterns, and different life-spans.This substantial variation means that it is useful to have aging clocksthat are specific to different cells, tissues, and organs (Seim, Ma, andGladyshev 2016). In a preferred embodiment we utilize DNN-basedpredictors of age trained on 12 tissues and 4 tissue-specific DNN-basedpredictors of age trained on gene expression profiles of a mononuclearwhole blood fraction.

Despite the fact that universal 12-tissues based predictor is trained atthe data set with a larger sample size compared to 4 tissues specificdeep aging clocks, its prediction performance is significantly worse(11.2 years for best network compared to 6.4, 8.2, 7.8 and 8.3 years forBlood, Brain, Liver and M. Blood-based predictors respectively).

In a preferred embodiment we utilize a DFS algorithm for feature rankingto identify the most important genes in age prediction on the universal12-tissues based predictor of age as well the 4 tissues specificpredictors of age.

In an implementation of the method a universal 12-tissues basedpredictor is trained on a data set with a larger sample size compared to4 tissues specific deep aging clocks, its prediction performance issignificantly worse (11.2 years for best network compared to 6.4, 8.2,7.8 and 8.3 years for Blood, Brain, Liver and M. Blood based predictors,respectively).

Data from up to 51,139 samples profiled on a GLP570 microarray platformwas used to train and test our DNNs. The GLP570 GEO accession numbersrefers to data generated using the common Affymetrix Human Genome U133Plus 2.0 Array, which covers approximately 47,000 transcripts, althoughonly 12,328 or 12,428 transcripts were used in the study. Data was splitinto training and test sets with a 90:10 ratio with exact values shownin each results section.

Following on from the successful and highly accurate usage of our DNN toclassify sex we then attempted to predict classify based on age ofsamples. As discussed previously we approached age prediction as aregression-based problem. In a preferred embodiment, 12,328 genes over atotal of 20,766 samples were used, 18,261 samples were used to train and2,505 samples used to test. Our DNN-based age predictor delivered a MAEof 11.46 years, a significant improvement over standard machine learningmodels, with k-NN coming closest to matching the DNN with a MAE of14.973 years. A very small increase (0.085) in MAE was observedfollowing DFS for the 1,000 most relevant genes suggesting that therewas little extra training capacity in the DNN using selected geneexpression dataset.

Since we saw a clear ability to distinguish tissues by our DNN weinvestigated if the MAE of the age predictor, would change wheninvestigating tissue specific aging. In a preferred embodiment, 12,428genes were analyzed from 1,853 samples from whole blood (1,733 train,120 test), 372 from brain (278 train, 49 test), 287 from liver (228train, 47 test) and 267 mononuclear blood fractions (170 train, 97test); again using a regression based model. Remarkably, in all cases asignificant improvement over the MAE of our general DNN-based agepredictor was observed, with whole blood performing especially wellgenerating a MAE of 6.696. Further improvements were seen following DFS,with a particularly large decrease in MAE observed in brain samples(10.788 vs 8.209). In all instances the various DNN outperformed RF,k-NN and LR models often producing an MAE more than 50% smaller. Intotal, these observations suggest that the transcriptomic aging-clock isregulated in a tissue specific manner.

Multilayer (with 3 or 4 hidden layers) feed-forward neural networks witha standard backpropagation algorithm were used in a preferredembodiment. A Python implementation of the Keras library with Theanobackend was used to build and train neural networks and Scikit-learnlibrary to build and train random forest (RF), K-nearest neighbor (k-NN)and linear regression (LR) models. Grid search algorithm was used forhyperparameter optimization in order to achieve the greatest predictiveaccuracy.

After rounds of optimization, Adam optimizer with Nesterov momentum andlearning rate of 0.01 was selected for all models. Rectified linear unit(ReLU) either exponential linear unit (ELU) were selected as activationfunctions. Mean absolute error (MAE) loss function was used in aregression task of age prediction. For regularization purposes modelswere trained with a dropout with 20-50% probability after each layer.Performance of the best DNNs were compared to best (with optimizedhyperparameters) RF and k-NN algorithms where appropriate. For thepurposes of this study we treated the prediction of human age as aregression based problem as previously discussed (Putin E 2017)therefore age related experiments are also compared against a LR model.All experiments were conducted with 5-fold cross validation by drugs onNVIDIA GTC Titan Pascal with 128 Gb of RAM.

Example 3

The first step of 5R strategy is rescuing pre-senescent cells in aparticular tissue (including skin, liver and lungs). Pre-senescentphenotype is considered potentially reversible. Senoremediators are thecompounds which can reverse the pre-senescent state in the tissue/organ.

Evidence indicates that BRAF(V600E) oncogene-induced senescence (OIS)can be reversed by activation of phosphatidylinositol 3-kinase (PI3K) orinhibition of PDH (Kaplon et al. 2013; Vredeveld et al. 2012).

Combined inhibition of both mTORC1 and mTORC2 also may provide apromising strategy to reverse the development of senescence-associatedfeatures in near-senescent cells (Walters, Deneka-Hannemann, and Cox2016).

In order to rescue the cells demonstrating pre-senescent phenotype thespecific set of possible interventions shall be applied. Theseinterventions include the treatment with the one senoremediator compoundor a combination of the senoremediator compounds from the list below.

Activators of PI3K: Insulin receptor substrate (Tyr608) peptide, thesequence is established and known in the art, is from insulin receptorsubstrate-1 (IRS-1) inclusive of Tyr608 (mouse)-Tyr612 (human). Itcontains the insulin receptor tyrosine kinase substrate motif YMXM(Tyr-Met-X-Met). This peptide has been used as a substrate for purifiedinsulin receptor (Km=90 μM) and other tyrosine kinases inphosphocellulose binding assays. The tyrosine phosphorylated version ofthis peptide binds to phosphatidylinositol 3-kinase (PI 3-kinase) SH2domain and activates the enzyme.

740 Y-P: cell-permeable phosphopeptide activator of PI3K. The PDGFR740Y-P peptide stimulates a mitogenic response in muscle cells. Theability of the 740Y-P peptide to stimulate mitogenesis is highlyspecific and not a general feature of a cell permeable SH2 domainbinding peptides. See ncbi.nlm.nih.gov/pubmed/9790922.

mTORC1, mTORC2 inhibitors: sapanisertib (Wise-Draper et al. 2017; Mooreet al. 2018), dactolisib (Wise-Draper et al. 2017).

Inhibitors of PDH: GSK2334470 (GlaxoSmithKline), MP7 (Merck).(Emmanouilidi and Falasca 2017).

Compounds found based on transcriptional signature analysis according tothe procedure described in example 1: Withaferin A, Lavendustin A,Sulforaphane.

Senoremediator compounds can be administered orally, by injection,sublingually, buccally, rectally, vaginally, cutaneously, transdermally,ocularly, oticly or nasally or other method.

Example 4

This step is performed to eliminate the cells that, already entered theirreversible senescent state. Senescent cells lose their function andpossess a constant danger to the surrounding cells as described above.Elimination of such cells may prevent surrounding cells to enter thesenescent phenotype by positive loop and restore the normal tissuefunctioning. In order to eliminate the cells demonstrating senescentphenotype the specific set of possible interventions shall be applied.These interventions include the treatment with the one senolyticcompound or a combination of the senolytic compounds from the listbelow.

Combination of dasatinib plus quercetin. As it was shown in (Xu et al.2018) combination of dasatinib plus quercetin of intermittent oraladministration to both senescent cell-transplanted young mice andnaturally aged mice alleviated physical dysfunction and increasedpost-treatment survival by 36% while reducing mortality hazard to 65%.

The following compounds have been found based on transcriptionalsignature analysis according to the procedure described in Example 1.

Flavopiridol, a flavonoid derived from an indigenous plant from India,demonstrated potent and specific in vitro inhibition of all cdks tested(cdks 1, 2, 4 and 7) with clear block in cell cycle progression at theG1/S and G2/M boundaries. Preclinical studies demonstrated the capacityof flavopiridol to induce programmed cell death, promotedifferentiation, inhibit angiogenic processes and modulatetranscriptional events (Senderowicz 1999).

Linifanib, an orally bioavailable, small-molecule receptor tyrosinekinase (RTK) inhibitor with potential antineoplastic activity. Linifanibinhibits members of the vascular endothelial growth factor (VEGF) andplatelet-derived growth factor (PDGF) receptor families; it exhibitsmuch less activity against unrelated RTKs, soluble tyrosine kinases, orserine/threonine kinases. This agent does not have a generalantiproliferative effect due to its high dose requirement. However,linifanib may exhibit potent antiproliferative and apoptotic effects ontumor cells whose proliferation is dependent on mutant kinases, such asfms-related tyrosine kinase receptor-3 (FLT3) (“NCI Drug Dictionary”n.d.).

Argatroban, is an anticoagulant that is a small molecule direct thrombininhibitor. (“Argatroban” n.d.).

Withaferin A, which is widely researched for its anti-inflammatory,cardioactive and central nervous system effects. (Mohan et al. 2004)

Senolytic compounds can be administered orally, by injection,sublingually (e.g., argatroban), buccally, rectally, vaginally,cutaneously, transdermally, ocularly, oticly or nasally or other method.

Example 5

The replenishment step leads to the general rejuvenation of the cells inthe population, but on the other hand, to the reduction in the totalcell count. This necessitate the further replenish step needed forrepopulation of the tissue with functional cells. Therefore the pool ofthe cells in a particular tissue should be activated in order toreplenish the tissue. The possible interventions needed to achieve thatgoal include the treatment with the one specific compound or acombination of the compounds from the list below.

It has been shown that senescent cells have been successfullydedifferentiated into pluripotent stem cells (Lapasset et al. 2011).

Transcription factors to turn cells into pluripotent: OSKM gene set(oct4, sox2, klf-4, myc), NANOG, Lin28 can be used in vitro to makepatient-specific pluripotent stem cells/different progenitors.

Importantly the compounds should stimulate the proliferation of the stemcells, but on the other hand prevent the unwanted effects related to thepossible uncontrolled proliferation and subsequent malignanttransformation. The compounds should be administered orally, byinjection, sublingually, buccally, rectally, vaginally, cutaneously,transdermally, ocularly, oticly or nasally or other method.

Example 6

The reinforcement step is required to prevent the further potentialdegradation of the tissue (or organ). It may include the treatment withthe one specific compound or a combination of the compounds from thelist below. These compounds should demonstrate one of the followingactivities: immunomodulation in order to prevent possible malignanttransformation and the accumulation of the senescent cells,cytoprotection in order to retain the functional state of the tissue,stimulation of the macrophages in order to achieve the specific state ofsenophagy (ability to specifically engulf and digest senescent cells).

Sorafenib inhibits tumor-cell proliferation and angiogenesis by blockingboth the intracellular Raf kinase pathway and extracellular VEGFRs andPDGFR-β associated kinases. (Abou-Alfa et al. 2017). Sorafenib has alsobeen reported to have immunomodulatory effects in addition to enhancingthe tyrosine kinase inhibition of cancer proliferation and angiogenesis.(Houben et al. 2009).

Tucaresol, an orally bioavailable immunopotentiatory drug, has beenshown to enhance T-helper-cell activity, with the induction of increasedIL-2 and IFN-γ levels in mice and humans (Rhodes et al. 1995).

Naturally occurring flavonoids discussed in the present article haveoptimal immunomodulation to prevent immune-mediated disorders throughmanagement of Th1/Th2 cytokine balance. (Gandhi et al. 2018).

Prevention of bleomycin-induced pulmonary fibrosis by IVIG (intravenousimmunoglobulin has been demonstrated by reduced expression of collagen-Iprotein in the affected lungs. (Molina et al. 2006).

Cell therapy with adipose tissue-derived stem cells (ASCs). Theimmunomodulatory and therapeutic effects of ASCs were confirmed in themouse model both in vitro and in vivo. (Yoshizumi et al. 2017).

Antigen independent immunomodulatory agents (like methotrexate,tacrolimus, and curcumin) delivered via transdermal route forimmunomodulation.

The compounds should be administered orally, by injection, sublingually,buccally, rectally, vaginally, cutaneously, transdermally, ocularly,oticly or nasally or other method.

The steps may be carried out in any order or repeated. A database whichcontains the full list is in (Aliper, Belikov, et al. 2016). This datacan be stored on a memory device in a computing system of the invention.

The stem cells may be mesenchymal, epithelial stem cells,induced-pluripotent stem cells or a combination. The method may furtherinclude applying a reinforcement drug treatment protocol to the patient,using, immunomodulation, cytoprotection, or stimulation of macrophages.

TABLE C Example list of small molecule molecules and short peptides forthe reinforcement step. Name Formula Insulin receptorenzolifesciences.com/bml-p320/irs-l-tyr608-peptide/ substrate (Tyr608)peptide 740 Y-P rndsystems.com/products/740-y-p_1983 Sapanisertib

Dactolisib

GSK2334470

MP7

Dasantinib

Quercetin

Flavopiridol

Linifanib

Argatroban

Sorafenib

Tucaresol

Methotrexate

Tacrolimus

Curcumin

Sulforaphane

Withaferin A

Lavendustin A

For these and other processes and methods disclosed herein, theoperations performed in the processes and methods may be implemented indiffering order. Furthermore, the outlined operations are only providedas examples, and some operations may be optional, combined into feweroperations, eliminated, supplemented with further operations, orexpanded into additional operations, without detracting from the essenceof the disclosed embodiments.

The present disclosure is not to be limited in terms of the particularembodiments described in this application, which are intended asillustrations of various aspects. Many modifications and variations canbe made without departing from its spirit and scope. Functionallyequivalent methods and apparatuses within the scope of the disclosure,in addition to those enumerated herein, are possible from the foregoingdescriptions. Such modifications and variations are intended to fallwithin the scope of the appended claims.

The present disclosure is to be limited only by the terms of theappended claims, along with the full scope of equivalents to which suchclaims are entitled. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

In one embodiment, the present methods can include aspects performed ona computing system. As such, the computing system can include a memorydevice that has the computer-executable instructions for performing themethods. The computer-executable instructions can be part of a computerprogram product that includes one or more algorithms for performing anyof the methods of any of the claims.

In one embodiment, any of the operations, processes, or methods,described herein can be performed or cause to be performed in responseto execution of computer-readable instructions stored on acomputer-readable medium and executable by one or more processors. Thecomputer-readable instructions can be executed by a processor of a widerange of computing systems from desktop computing systems, portablecomputing systems, tablet computing systems, hand-held computingsystems, as well as network elements, and/or any other computing device.The computer readable medium is not transitory. The computer readablemedium is a physical medium having the computer-readable instructionsstored therein so as to be physically readable from the physical mediumby the computer/processor.

There are various vehicles by which processes and/or systems and/orother technologies described herein can be effected (e.g., hardware,software, and/or firmware), and that the preferred vehicle may vary withthe context in which the processes and/or systems and/or othertechnologies are deployed. For example, if an implementer determinesthat speed and accuracy are paramount, the implementer may opt for amainly hardware and/or firmware vehicle; if flexibility is paramount,the implementer may opt for a mainly software implementation; or, yetagain alternatively, the implementer may opt for some combination ofhardware, software, and/or firmware.

The various operations described herein can be implemented, individuallyand/or collectively, by a wide range of hardware, software, firmware, orvirtually any combination thereof. In one embodiment, several portionsof the subject matter described herein may be implemented viaapplication specific integrated circuits (ASICs), field programmablegate arrays (FPGAs), digital signal processors (DSPs), or otherintegrated formats. However, some aspects of the embodiments disclosedherein, in whole or in part, can be equivalently implemented inintegrated circuits, as one or more computer programs running on one ormore computers (e.g., as one or more programs running on one or morecomputer systems), as one or more programs running on one or moreprocessors (e.g., as one or more programs running on one or moremicroprocessors), as firmware, or as virtually any combination thereof,and that designing the circuitry and/or writing the code for thesoftware and/or firmware are possible in light of this disclosure. Inaddition, the mechanisms of the subject matter described herein arecapable of being distributed as a program product in a variety of forms,and that an illustrative embodiment of the subject matter describedherein applies regardless of the particular type of signal bearingmedium used to actually carry out the distribution. Examples of aphysical signal bearing medium include, but are not limited to, thefollowing: a recordable type medium such as a floppy disk, a hard diskdrive (HDD), a compact disc (CD), a digital versatile disc (DVD), adigital tape, a computer memory, or any other physical medium that isnot transitory or a transmission. Examples of physical media havingcomputer-readable instructions omit transitory or transmission typemedia such as a digital and/or an analog communication medium (e.g., afiber optic cable, a waveguide, a wired communication link, a wirelesscommunication link, etc.).

It is common to describe devices and/or processes in the fashion setforth herein, and thereafter use engineering practices to integrate suchdescribed devices and/or processes into data processing systems. Thatis, at least a portion of the devices and/or processes described hereincan be integrated into a data processing system via a reasonable amountof experimentation. A typical data processing system generally includesone or more of a system unit housing, a video display device, a memorysuch as volatile and non-volatile memory, processors such asmicroprocessors and digital signal processors, computational entitiessuch as operating systems, drivers, graphical user interfaces, andapplications programs, one or more interaction devices, such as a touchpad or screen, and/or control systems, including feedback loops andcontrol motors (e.g., feedback for sensing position and/or velocity;control motors for moving and/or adjusting components and/orquantities). A typical data processing system may be implementedutilizing any suitable commercially available components, such as thosegenerally found in data computing/communication and/or networkcomputing/communication systems.

The herein described subject matter sometimes illustrates differentcomponents contained within, or connected with, different othercomponents. Such depicted architectures are merely exemplary, and thatin fact, many other architectures can be implemented which achieve thesame functionality. In a conceptual sense, any arrangement of componentsto achieve the same functionality is effectively “associated” such thatthe desired functionality is achieved. Hence, any two components hereincombined to achieve a particular functionality can be seen as“associated with” each other such that the desired functionality isachieved, irrespective of architectures or intermedial components.Likewise, any two components so associated can also be viewed as being“operably connected”, or “operably coupled”, to each other to achievethe desired functionality, and any two components capable of being soassociated can also be viewed as being “operably couplable”, to eachother to achieve the desired functionality. Specific examples ofoperably couplable include, but are not limited to: physically mateableand/or physically interacting components and/or wirelessly interactableand/or wirelessly interacting components and/or logically interactingand/or logically interactable components.

Unless specifically stated otherwise, as apparent from the followingdiscussions, it is appreciated that throughout the specificationdiscussions utilizing terms such as “processing”, “computing”,“calculating”, “determining”, “deriving”, “generating” or the like,refer to the action and/or processes of a computer or computing system,or processor or similar electronic computing device, that manipulateand/or transform data represented as physical, such as electronic,quantities within the computing system's registers and/or memories intoother data, similarly represented as physical quantities within thecomputing system's memories, registers or other such informationstorage, transmission or display devices.

It is noteworthy that any hardware platform suitable for performing theprocessing described herein is suitable for use with the systems andmethods provided herein. Computer-readable storage media refer to anymedium or media that participate in providing instructions to a centralprocessing unit (CPU), a processor, a microcontroller, or the like. Suchmedia may take forms including, but not limited to, non-volatile andvolatile media such as optical or magnetic disks and dynamic memory,respectively. Common forms of computer-readable storage media include afloppy disk, a flexible disk, a hard disk, magnetic tape, any othermagnetic storage medium, a CD-ROM disk, digital video disk (DVD), anyother optical storage medium, RAM, PROM, EPROM, a FLASHEPROM, any othermemory chip or cartridge.

Computer program code for carrying out operations for aspects of thepresent technology may be written in any combination of one or moreprogramming languages, including an object oriented programming languagesuch as Python, Java, Smalltalk, C++ or the like and conventionalprocedural programming languages, such as the “C” programming languageor similar programming languages. The program code may execute entirelyon the user's computer, partly on the user's computer, as a stand-alonesoftware package, partly on the user's computer and partly on a remotecomputer or entirely on the remote computer or server. In the latterscenario, the remote computer may be coupled with the user's computerthrough any type of network, including a local area network (LAN) or awide area network (WAN), or the connection may be made to an externalcomputer (for example, through the Internet using an Internet ServiceProvider).

FIG. 12 shows an example computing device 600 (e.g., a computer) thatmay be arranged in some embodiments to perform the methods (or portionsthereof) described herein. In a very basic configuration 602, computingdevice 600 generally includes one or more processors 604 and a systemmemory 606. A memory bus 608 may be used for communicating betweenprocessor 604 and system memory 606.

Depending on the desired configuration, processor 604 may be of any typeincluding, but not limited to: a microprocessor (μP), a microcontroller(μC), a digital signal processor (DSP), or any combination thereof.Processor 604 may include one or more levels of caching, such as a levelone cache 610 and a level two cache 612, a processor core 614, andregisters 616. An example processor core 614 may include an arithmeticlogic unit (ALU), a floating point unit (FPU), a digital signalprocessing core (DSP Core), or any combination thereof. An examplememory controller 618 may also be used with processor 604, or in someimplementations, memory controller 618 may be an internal part ofprocessor 604.

Depending on the desired configuration, system memory 606 may be of anytype including, but not limited to: volatile memory (such as RAM),non-volatile memory (such as ROM, flash memory, etc.), or anycombination thereof. System memory 606 may include an operating system620, one or more applications 622, and program data 624. Application 622may include a determination application 626 that is arranged to performthe operations as described herein, including those described withrespect to methods described herein. The determination application 626can obtain data, such as pressure, flow rate, and/or temperature, andthen determine a change to the system to change the pressure, flow rate,and/or temperature.

Computing device 600 may have additional features or functionality, andadditional interfaces to facilitate communications between basicconfiguration 602 and any required devices and interfaces. For example,a bus/interface controller 630 may be used to facilitate communicationsbetween basic configuration 602 and one or more data storage devices 632via a storage interface bus 634. Data storage devices 632 may beremovable storage devices 636, non-removable storage devices 638, or acombination thereof. Examples of removable storage and non-removablestorage devices include: magnetic disk devices such as flexible diskdrives and hard-disk drives (HDD), optical disk drives such as compactdisk (CD) drives or digital versatile disk (DVD) drives, solid statedrives (SSD), and tape drives to name a few. Example computer storagemedia may include: volatile and non-volatile, removable andnon-removable media implemented in any method or technology for storageof information, such as computer readable instructions, data structures,program modules, or other data.

System memory 606, removable storage devices 636 and non-removablestorage devices 638 are examples of computer storage media. Computerstorage media includes, but is not limited to: RAM, ROM, EEPROM, flashmemory or other memory technology, CD-ROM, digital versatile disks (DVD)or other optical storage, magnetic cassettes, magnetic tape, magneticdisk storage or other magnetic storage devices, or any other mediumwhich may be used to store the desired information and which may beaccessed by computing device 600. Any such computer storage media may bepart of computing device 600.

Computing device 600 may also include an interface bus 640 forfacilitating communication from various interface devices (e.g., outputdevices 642, peripheral interfaces 644, and communication devices 646)to basic configuration 602 via bus/interface controller 630. Exampleoutput devices 642 include a graphics processing unit 648 and an audioprocessing unit 650, which may be configured to communicate to variousexternal devices such as a display or speakers via one or more A/V ports652. Example peripheral interfaces 644 include a serial interfacecontroller 654 or a parallel interface controller 656, which may beconfigured to communicate with external devices such as input devices(e.g., keyboard, mouse, pen, voice input device, touch input device,etc.) or other peripheral devices (e.g., printer, scanner, etc.) via oneor more I/O ports 658. An example communication device 646 includes anetwork controller 660, which may be arranged to facilitatecommunications with one or more other computing devices 662 over anetwork communication link via one or more communication ports 664.

The network communication link may be one example of a communicationmedia. Communication media may generally be embodied by computerreadable instructions, data structures, program modules, or other datain a modulated data signal, such as a carrier wave or other transportmechanism, and may include any information delivery media. A “modulateddata signal” may be a signal that has one or more of its characteristicsset or changed in such a manner as to encode information in the signal.By way of example, and not limitation, communication media may includewired media such as a wired network or direct-wired connection, andwireless media such as acoustic, radio frequency (RF), microwave,infrared (IR), and other wireless media. The term computer readablemedia as used herein may include both storage media and communicationmedia.

Computing device 600 may be implemented as a portion of a small-formfactor portable (or mobile) electronic device such as a cell phone, apersonal data assistant (PDA), a personal media player device, awireless web-watch device, a personal headset device, an applicationspecific device, or a hybrid device that includes any of the abovefunctions. Computing device 600 may also be implemented as a personalcomputer including both laptop computer and non-laptop computerconfigurations. The computing device 600 can also be any type of networkcomputing device. The computing device 600 can also be an automatedsystem as described herein.

The embodiments described herein may include the use of a specialpurpose or general-purpose computer including various computer hardwareor software modules.

Embodiments within the scope of the present invention also includecomputer-readable media for carrying or having computer-executableinstructions or data structures stored thereon. Such computer-readablemedia can be any available media that can be accessed by a generalpurpose or special purpose computer. By way of example, and notlimitation, such computer-readable media can comprise RAM, ROM, EEPROM,CD-ROM or other optical disk storage, magnetic disk storage or othermagnetic storage devices, or any other medium which can be used to carryor store desired program code means in the form of computer-executableinstructions or data structures and which can be accessed by a generalpurpose or special purpose computer. When information is transferred orprovided over a network or another communications connection (eitherhardwired, wireless, or a combination of hardwired or wireless) to acomputer, the computer properly views the connection as acomputer-readable medium. Thus, any such connection is properly termed acomputer-readable medium. Combinations of the above should also beincluded within the scope of computer-readable media.

Computer-executable instructions comprise, for example, instructions anddata which cause a general purpose computer, special purpose computer,or special purpose processing device to perform a certain function orgroup of functions. Although the subject matter has been described inlanguage specific to structural features and/or methodological acts, itis to be understood that the subject matter defined in the appendedclaims is not necessarily limited to the specific features or actsdescribed above. Rather, the specific features and acts described aboveare disclosed as example forms of implementing the claims.

With respect to the use of substantially any plural and/or singularterms herein, those having skill in the art can translate from theplural to the singular and/or from the singular to the plural as isappropriate to the context and/or application. The varioussingular/plural permutations may be expressly set forth herein for sakeof clarity.

It will be understood by those within the art that, in general, termsused herein, and especially in the appended claims (e.g., bodies of theappended claims) are generally intended as “open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.). It will be further understood by those within the art that if aspecific number of an introduced claim recitation is intended, such anintent will be explicitly recited in the claim, and in the absence ofsuch recitation, no such intent is present. For example, as an aid tounderstanding, the following appended claims may contain usage of theintroductory phrases “at least one” and “one or more” to introduce claimrecitations. However, the use of such phrases should not be construed toimply that the introduction of a claim recitation by the indefinitearticles “a” or “an” limits any particular claim containing suchintroduced claim recitation to embodiments containing only one suchrecitation, even when the same claim includes the introductory phrases“one or more” or “at least one” and indefinite articles such as “a” or“an” (e.g., “a” and/or “an” should be interpreted to mean “at least one”or “one or more”); the same holds true for the use of definite articlesused to introduce claim recitations. In addition, even if a specificnumber of an introduced claim recitation is explicitly recited, thoseskilled in the art will recognize that such recitation should beinterpreted to mean at least the recited number (e.g., the barerecitation of “two recitations,” without other modifiers, means at leasttwo recitations, or two or more recitations). Furthermore, in thoseinstances where a convention analogous to “at least one of A, B, and C,etc.” is used, in general, such a construction is intended in the senseone having skill in the art would understand the convention (e.g., “asystem having at least one of A, B, and C” would include but not belimited to systems that have A alone, B alone, C alone, A and Btogether, A and C together, B and C together, and/or A, B, and Ctogether, etc.). It will be further understood by those within the artthat virtually any disjunctive word and/or phrase presenting two or morealternative terms, whether in the description, claims, or drawings,should be understood to contemplate the possibilities of including oneof the terms, either of the terms, or both terms. For example, thephrase “A or B” will be understood to include the possibilities of “A”or “B” or “A and B.”

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and allpurposes, such as in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” and the like include the number recited andrefer to ranges which can be subsequently broken down into subranges asdiscussed above. Finally, as will be understood by one skilled in theart, a range includes each individual member. Thus, for example, a grouphaving 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, agroup having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells,and so forth.

From the foregoing, it will be appreciated that various embodiments ofthe present disclosure have been described herein for purposes ofillustration, and that various modifications may be made withoutdeparting from the scope and spirit of the present disclosure.Accordingly, the various embodiments disclosed herein are not intendedto be limiting, with the true scope and spirit being indicated by thefollowing claims.

Definitions

A “biopsy” is a medical test involving extraction of sample cells ortissues for examination, and can be analyzed chemically. When only asample of tissue is removed with preservation of the histologicalarchitecture of the tissue's cells, the procedure is called anincisional biopsy or core biopsy. When a sample of tissue or fluid isremoved with a needle in such a way that cells are removed withoutpreserving the histological architecture of the tissue cells, theprocedure is called a needle aspiration biopsy.

“Senescence” is biological aging, that is, the gradual deterioration offunction and ability in almost all life forms, mostly after maturationand in particular multi-cellular life. Senescence increases mortality.Senescence refer to cellular senescence, tissue senescence, organsenescence, and senescence of the whole organism. Cellular senescencelargely underlies organismal senescence. The boundary between diseaseand senescence as organisms, tissues, and cells, may havecharacteristics of both, as disease and senescence are often associatedwith each other.

“Cellular senescence” is not the aging of an individual cell, butinstead, the state (gene expression) of a cell with respect to thesenescence of its tissue or organism, in comparison to a less senescenttissue or organism. Cell senescence may partly be the result of telomereshortening cells, which may trigger a DNA damage response. Cells canalso be induced to senesce via DNA damage in response to elevatedreactive oxygen species, activation of oncogenes, cell-to-cell fusion,and other causes. As such, cellular senescence represents a change in“cell state” rather than a cell becoming “aged” The number of senescentcells in tissues rises substantially during normal aging. Cells may alsoexperience “replicative senescence”, in which they can no longer divide.There is a “senescence associated secretory phenotype” (SASP) associatedwith senescent cells, which is associated with, for example, an increasein inflammatory cytokines, growth factors, and proteases. Cellularsenescence contributes to age-related diseases, such as atherosclerosis.

“Fibrosis” is the accumulation of excess fibrous connective cells orother similarly stiff, structural cells, called “fibrotic cells” in anorgan or tissue. Such fibrosis can be a normal, functional part of thereparative process (such as scarring) but can also be pathological.Excess and unnecessary fibrosis is associated with senescence, typicallydecrease flexibility and other function of a tissue or organ. Fibroticcells generally have an excess of extracellular matrix proteins whichcontribute to their stiffness.

A “senolytic” is a drug of other treatment that can selectively inducedeath of senescent cells.

A “senoremediator” is a drug of other treatment that can restore orincrease the number of presenescent or nonsenescent cells.

“Machine learning” (ML) is a subfield of computer science that givescomputers the ability to learn without being explicitly programmed.Machine learning platforms include, but are not limited to naïve bayesclassifiers, support vector machines, decision trees, and neuralnetworks.

“Artificial neural networks”, also called “ANNs” or just “neuralnetworks”, are based on a large collection of connected simple unitscalled artificial neurons loosely analogous to axons in a biologicalbrain. If the combined incoming signals are strong enough, the neuronbecomes activated and the signal travels to other neurons connected toit. The activation function of such neurons is often, though not always,represented as a sigmoid function.

“Deep learning” (DL) (also known as deep structured learning,hierarchical learning or deep machine learning) is the study ofartificial neural networks that contain more than one hidden layer ofneurons. Such a neural network is called a “deep neural network”. A“convolutional neural network” is a type of neural network in which theconnectivity pattern is inspired by the organization of the animalvisual cortex.

“Principal component analysis” (PCA) is a statistical procedure thatuses an orthogonal transformation to convert a set of observations ofvariables into a set of values of linearly uncorrelated variables calledprincipal components. The transformation is defined in such a way thatthe first principal component has the largest possible variance and eachsucceeding component in turn has the highest variance possible under theconstraint that it is orthogonal to the preceding components.

“Generative adversarial networks” (GANs) are neural networks that aretrained in an adversarial manner to generate data mimicking somedistribution. A discriminative model is a model that discriminatesbetween two (or more) different classes of data, for example aconvolutional neural network that is trained to output 1 given an imageof a human face and 0 otherwise. A generative model by contrastgenerates new data which fits the distribution of the training data.GANs are well known in the art, as described, for example, in (2)Goodfellow et. al., “Generative Adversarial Networks”,arXiv:1406.2661v1, 2014.

An “autoencoder” is a neural network architecture generally used forunsupervised learning of efficient coding. An autoencoder learnrepresentations (encodings) for a set of data, often for the purpose ofdimensionality reduction. An “adversarial autoencoder” (AAE), is anautoencoder that uses generative adversarial networks (GAN) to performvariational inference by matching the aggregated posterior of the hiddencode vector of the autoencoder with an arbitrary prior distribution.AAEs are well known in the art, as described, for example, in (3)Makhzani et. al., “Adversarial Autoencoders”, arXiv:1511.05644v2, 2015.Application of AAEs to new molecule development such as drugs is alsowell-known in the art, as described, for example, in (4) Kadurin, et.al., “The cornucopia of meaningful leads: Applying deep adversarialautoencoders for new molecule development in oncology”, Oncotarget,2017, Vol. 8, (No. 7), pp: 10883-10890.

All references recited herein and/or recited in the provisionalapplications 62/536,658 filed Jul. 25, 2017 and/or 62/547,061 filed Aug.17, 2017 are incorporated herein by specific reference in theirentirety.

The following References are background literature for operation of thetechnology described herein, and are incorporated herein by specificreference.

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1. A method of treating senescence in a subject, the method comprising:applying a senoremediation drug treatment protocol to the subject inorder to rescue one or more first cells in the subject, wherein thesenoremediation drug treatment protocol is derived from a computationaltranscriptome analysis of the tissue or organ of the subject.
 2. Themethod of claim 1, further comprising applying a senolytic drugtreatment protocol to the subject in order to remove one or more secondcells in the subject.
 3. The method of claim 2, wherein the senolyticdrug treatment protocol is derived from the computational transcriptomeanalysis of the tissue or organ of the subject.
 4. The method of claim1, further comprising introducing stem cells into a tissue and/or organof the subject in order to rejuvenate one or more tissue cells in thetissue and/or one or more organ cells in the organ.
 5. The method ofclaim 4, wherein at least one of the following is derived from acomputational transcriptome analysis of the tissue or organ of thesubject: identification of the tissue for receiving the stem cells; oridentification of the organ for receiving the stem cells.
 6. The methodof claim 1, further comprising carrying out a reinforcement step thatincludes one or more actions that prevent further senescence ordegradation of the tissue or organ.
 7. The method of claim 6, whereinthe one or more actions that prevent further senescence or degradationof the tissue or organ is derived from the computational transcriptomeanalysis of the tissue or organ of the subject.
 8. The method of claim1, further comprising: applying a senolytic drug treatment protocol tothe subject in order to remove one or more second cells in the subject;and introducing stem cells into a tissue and/or organ of the subject inorder to rejuvenate one or more tissue cells in the tissue and/or one ormore organ cells in the organ.
 9. The method of claim 8, wherein atleast one of the following is derived from a computational transcriptomeanalysis of the tissue or organ of the subject: the senolytic drugtreatment protocol; identification of the tissue for receiving the stemcells; or identification of the organ for receiving the stem cells. 10.The method of claim 8, further comprising carrying out a reinforcementstep that includes one or more actions that prevent further senescenceor degradation of the tissue or organ.
 11. The method of claim 10,wherein at least one of the following is derived from a computationaltranscriptome analysis of the tissue or organ of the subject: thesenolytic drug treatment protocol; identification of the tissue forreceiving the stem cells; identification of the organ for receiving thestem cells; or the one or more actions that prevent further senescenceor degradation of the tissue or organ.
 12. A method of treatingsenescence in a subject, the method comprising: (a) applying asenoremediation drug treatment protocol to the subject in order torescue one or more first cells in the subject; (b) applying a senolyticdrug treatment protocol to the subject in order to remove one or moresecond cells in the subject; (c) introducing stem cells into a tissueand/or organ of the subject in order to rejuvenate one or more tissuecells in the tissue and/or one or more organ cells in the organ; and (d)carrying out a reinforcement step that includes one or more actions thatprevent further senescence or degradation of the tissue or organ. 13.The method of claim 12, wherein at least one of the following is derivedfrom a computational transcriptome analysis of the tissue or organ ofthe subject: the senoremediation drug treatment protocol; the senolyticdrug treatment protocol; identification of the tissue for receiving thestem cells; identification of the organ for receiving the stem cells; orthe one or more actions that prevent further senescence or degradationof the tissue or organ.
 14. The method of claim 12, wherein the firstcells that are rescued are characterized as pre-senescent cells.
 15. Themethod of claim 12, wherein the second cells that are removed arecharacterized as senescent cells.
 16. The method of claim 12, wherein(a) (b) (c) or (d) are carried out in any order or any combinationthereof carried out simultaneously.
 17. The method of claim 12, furthercomprising repeating at least one of (a) (b) (c) or (d) at least once.18. The method of claim 12, wherein the stem cells are mesenchymal orepithelial stem cells or both.
 19. The method of claim 12, wherein step(d) further comprises applying a reinforcement drug treatment protocolto the patient.
 20. The method of claim 19, wherein the reinforcementdrug treatment protocol includes at least one of: immunomodulation,cytoprotection, or stimulation of macrophages.
 21. The method of claim19, wherein the reinforcement drug treatment protocol includes at leastone of: Insulin receptor substrate (Tyr608) peptide; 740 Y-P;Sapanisertib; Dactolisib; GSK2334470; MP7; Dasantinib; Quercitin;Flavopiridol; Linifanib; Argatroban; Sorafenib; Tucaresol; Methotrexate;Tacrolimus; Curcumin; Withaferin A; Sulphoraphane; Lavendustin A. 22.The method of claim 13, wherein the computational transcriptome analysisproduces a transcriptome signature that is used as input to a machinelearning platform that outputs drug classifications for one of steps(a), (b), or (d).
 23. The method of claim 13, wherein the computationaltranscriptome analysis produces a transcriptome signature that iscompared to a baseline transcriptome signature that represents a lesssenescent version of the subject's tissue or organ.
 24. The method ofclaim 13, wherein the computational transcriptome analysis produces atranscriptome signature that is compared to a baseline transcriptomesignature that is constructed from more than one tissue or organtranscriptome signature of one or more different subjects.